Factor H binding protein variants and methods of use thereof

ABSTRACT

Variant factor H binding proteins that can elicit antibodies that are bactericidal for at least one strain of Neisseria meningitidis, compositions comprising such proteins, and methods of use of such proteins, are provided.

CROSS-REFERENCE

This application is a continuation of 16/288,760 filed on Feb. 28, 2019,now U.S. Pat. No. 10,487,122; which is a divisional of U.S. applicationSer. No. 15/327,346 filed Jan. 18, 2017, now U.S. Pat. No. 10,266,572;which is a national phase of PCT/US2015/041616 filed on Jul. 22, 2015;which claims the benefit of U.S. Provisional Patent Application No.62/028,123, filed Jul. 23, 2014, which application is incorporatedherein by reference in its entirety.

INTRODUCTION

Neisseria meningitidis is a Gram-negative bacterium that colonizes thehuman upper respiratory tract and is responsible for worldwide sporadicand cyclical epidemic outbreaks of, most notably, meningitis and sepsis.The attack and morbidity rates are highest in children under 2 years ofage. Like other Gram-negative bacteria, Neisseria meningitidis typicallypossess a cytoplasmic membrane, a peptidoglycan layer, an outermembrane, which together with the capsular polysaccharide constitute thebacterial wall, and pili, which project into the outside environment.Encapsulated strains of Neisseria meningitidis are a major cause ofbacterial meningitis and septicemia in children and young adults. Theprevalence and economic importance of invasive Neisseria meningitidisinfections have driven the search for effective vaccines that can conferimmunity across different strains, and particularly across geneticallydiverse serogroup B strains with different serotypes or serosubtypes.

Factor H Binding Protein (fHbp, also referred to in the art aslipoprotein 2086 (Fletcher et al (2004) Infect Immun 72:2088-2100),Genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. (2003) JExp Med 197:789-99) or “741”) is an N. meningitidis protein that isexpressed in the bacterium as a surface-exposed lipoprotein. Animportant function of fHbp is to bind human complement factor H (fH),which down-regulates complement activation. Binding of fH to thebacterial surface is an important mechanism by which the pathogensurvives in non-immune human serum or blood and evades innate hostdefenses. Recently, genetic variation in the human factor H gene clusterwas found to affect susceptibility to developing meningococcal disease(Davila S et al. (2010) Nat Genetics doi:10.1038/ng.640). Binding of fHto fHbp is specific for human fH, and several non-human primates andcould partially explain why Neisseria meningitidis is strictly a humanpathogen. fHbp occurs in many natural sequence variants that aredesignated by identification (ID) numbers as assigned in the fHbpdatabase on the internet at pubmlst(dot)org/neisseria/fHbp.

There remains a need for an fHbp polypeptide that can elicit effectivebactericidal antibody responses.

SUMMARY

Variant factor H binding proteins that can elicit antibodies that arebactericidal for at least one strain of Neisseria meningitidis,compositions comprising such proteins, and methods of use of suchproteins, are provided.

FEATURES

The present disclosure provides variants of factor H binding protein(fHbp) ID 1. The present disclosure provides a variant of fHbp whereinthe variant comprises an amino acid substitution selected from at leastone of: a) an amino acid substitution of the glutamine at amino acid 38(Q38); b) an amino acid substitution of the glutamic acid at amino acid92 (E92); c) a substitution of glycine for arginine at amino acid 130(R130G); d) an amino acid substitution of the serine at amino acid 223(S223); and e) a substitution of histidine for leucine at amino acid 248(H248L), wherein the amino acid substitutions are relative to fHbp ID 1(SEQ ID NO:1), wherein the variant comprises an amino acid sequencehaving at least 80% amino acid sequence identity to SEQ ID NO:1, whereinthe variant fHbp binds human factor H (fH) with an affinity that is 50%or less of the affinity of fHbp ID 1 for human fH, and wherein thevariant induces a bactericidal antibody response to at least one strainof Neisseria meningitidis in a mammalian host. In some cases, the aminoacid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. Insome cases, the amino acid substitution at E92 is E92K, E92R, E92H,E92F, E92Y, or E92W. In some cases, the amino acid substitution at S223is S223R, S223K, S223H, S223F, S223Y, or S223W. In some cases, thevariant fHbp may further include a R41S or a R41A substitution relativeto fHbp ID 1. For example the variant fHbp may include a R41S or a R41Asubstitution and a substitution at S223, e.g., R41S/S223R, relative tofHbp ID 1. In other cases, the variant fHbp may further include a R41Sor a R41A substitution and a H248L substitution relative to fHbp ID 1.In certain cases, the variant fHbp may include two, three, or more ofthe substitutions disclosed herein. In a specific example, the variantfHbp may include the following substitutions: S223R and H248L relativeto fHbp ID 1. In some cases, the variant fHbp binds human fH with anaffinity that is 25% or less of the affinity of the fHbp ID 1 for humanfH. In some cases, the variant fHbp binds human fH with an affinity thatis 10% or less of the affinity of the fHbp ID 1 for human fH. In somecases, the variant fHbp binds human fH with an affinity that is 5% orless of the affinity of the fHbp ID 1 for human fH.

The present disclosure provides variants of fHbp ID 22. The presentdisclosure provides a variant of fHbp, wherein the variant comprises atleast one amino acid substitution selected from: a) a substitution ofisoleucine for asparagine at amino acid 115 (N115I); b) a substitutionof glycine for aspartic acid at amino acid 121 (D121G); c) asubstitution of threonine for serine at amino acid 128 (S128T); d) anamino acid substitution of the valine at position 131 (V131); e) anamino acid substitution of the lysine at position 219 (K219); f) anamino acid substitution of the glycine at position 220 (G220), whereinthe amino acid substitutions are relative to fHbp ID 22 (SEQ ID NO:2),wherein the variant comprises an amino acid sequence having greater than85% amino acid sequence identity to SEQ ID NO:2, wherein the variantfHbp binds human factor H (fH) with an affinity that is 50% or less ofthe affinity of fHbp ID 22 for human fH, and wherein the variant inducesa bactericidal antibody response in a mammalian host. In some cases, thevariant fHbp binds human fH with an affinity that is 25% or less of theaffinity of the fHbp ID 22 for human fH. In some cases, the variant fHbpbinds human fH with an affinity that is 10% or less of the affinity ofthe fHbp ID 22 for human fH. In some cases, the variant fHbp binds humanfH with an affinity that is 5% or less of the affinity of the fHbp ID 22for human fH. In some cases, the amino acid substitution at V131 isV131D, V131E, V131K, V131R, V131H, V131F, V131Y, or V131W. In somecases, the amino acid substitution at K219 is K219N, K219Q, K219D,K219E, K219F, K219Y, or K219W. In some cases, the amino acidsubstitution at G220 is G220S, G220N, G220Q, G220D, G220E, G220K, G220R,G220H, G220F, G220Y, or G220W.

In some cases, the variant fHbp includes a double mutation thatincreases thermal stability of the variant fHbp compared to thermalstability of wild type (WT) fHbp, e.g., WT fHbp ID 22. In some cases,the variant fHbp may include the substitutions L130R and G133D relativeto fHbp ID 22 (SEQ ID NO:2), wherein the variant fHbp comprises an aminoacid sequence having greater than 85% amino acid sequence identity toSEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with anaffinity that is 50% or less of the affinity of fHbp ID 22 for human fH,wherein the variant induces a bactericidal antibody response in amammalian host, and wherein the variant has a higher thermal stabilitycompared to thermal stability of fHbp ID 22. In some cases, the variantfHbp may include a combination of substitutions, such as, L130R, G133D,and at least one amino acid substitution selected from: a) N115I; b)D121G; c) S128T; d) V131; e) K219 (e.g., K219N); and f) G220 (e.g.,G220S), wherein the amino acid substitutions are relative to fHbp ID 22(SEQ ID NO:2), wherein the variant fHbp comprises an amino acid sequencehaving greater than 85% amino acid sequence identity to SEQ ID NO:2,wherein the variant fHbp binds human factor H (fH) with an affinity thatis 50% or less of the affinity of fHbp ID 22 for human fH, and whereinthe variant induces a bactericidal antibody response in a mammalianhost. The thermal stability of the variant fHbp may be higher than a WTfHbp (e.g., fHbp ID 22) by at least 5° C., 10° C., 15° C., 20° C., ormore, e.g., higher by 5° C.-30° C., 5° C.-25° C., 5° C.-20° C., 10°C.-20° C., or 15° C.-20° C. As used herein, “thermal stability” refersto stability of a protein when exposed to higher temperature; a thermalstability variant protein maintains its conformation at a highertemperature than a wild type protein. For example, the variant fHbp,that include the double mutation that increases thermal stabilitycompared to thermal stability of wild type (WT) fHbp, e.g., WT fHbp ID22, may unfold at a higher temperature compared to WT fHbp. In certaincases, the N-terminal domain of the variant fHbp may unfold at a highertemperature than the N-terminal domain of the WT fHbp (e.g., fHbp ID22).

Also disclosed herein are fHbp variants that include the mutations thatenhance thermal stability as compared to a WT fHbp and further includeadditional mutations known to reduce binding of fH, such as, thosedisclosed in US2011/0256180. In certain embodiments, a variant of factorH binding protein (fHbp) is disclosed, wherein the variant comprisesamino acid substitutions L130R and G133D and at least one of thesubstitutions: R80A, D211A, E218A, E248A, G236I, T221A, and H223Arelative to fHbp ID 22 (SEQ ID NO:2), wherein the variant comprises anamino acid sequence having greater than 85% amino acid sequence identityto SEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) withan affinity that is 50% or less of the affinity of fHbp ID 22 for humanfH, and wherein the variant induces a bactericidal antibody response ina mammalian host.

The present disclosure provides variants of fHbp ID 55. The presentdisclosure provides a variant of fHbp, wherein the variant comprises atleast one amino acid substitution selected from the group consisting of:a) an amino acid substitution of the glutamic acid at position 92 (E92);b) an amino acid substitution of the serine at position 223 (S223); andc) an amino acid substitution of the histidine at position 248 (H248),wherein the amino acid substitutions are relative to fHbp ID 55 (SEQ IDNO:3), wherein the variant comprises an amino acid sequence having atleast 90% amino acid sequence identity to SEQ ID NO:3, wherein thevariant fHbp binds human factor H (fH) with an affinity that is lessthan 50% of the affinity of fHbp ID 55 for human fH, and wherein thevariant induces a bactericidal antibody response in a mammalian host. Insome cases, the variant fHbp binds human fH with an affinity that is 25%or less of the affinity of the fHbp ID 55 for human fH. In some cases,the variant fHbp binds human fH with an affinity that is 10% or less ofthe affinity of the fHbp ID 55 for human fH. In some cases, the variantfHbp binds human fH with an affinity that is 5% or less of the affinityof the fHbp ID 55 for human fH. In some cases, the amino acidsubstitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In somecases, the amino acid substitution at S223 is S223R, S223K, S223H,S223F, S223Y, or S223W. In some cases, the amino acid substitution atH248 is H248L, H248I, H248V, H248D, H248E, H248F, H248Y, or H248W.

The present disclosure provides immunogenic compositions comprising avariant fHbp of the present disclosure. The present disclosure providesan immunogenic composition comprising: a) the variant fHbp according toany one of paragraphs 0006-0010 above; and b) a pharmaceuticallyacceptable excipient. In some cases, the fHbp variant is in a vesiclepreparation prepared from a Neisseria meningitidis strain. In somecases, the pharmaceutically acceptable excipient comprises an adjuvant;e.g., where the adjuvant is aluminum phosphate or aluminum hydroxide. Insome cases, the pharmaceutical composition further includes Neisserialsurface protein A.

The present disclosure provides a nucleic acid encoding a variant fHbpaccording to any one of paragraphs 0006-0010 above. The presentdisclosure provides a recombinant expression vector comprising a nucleicacid encoding a variant fHbp according to any one of paragraphs0006-0010 above. The present disclosure provides an in vitro host cellcomprising a nucleic acid encoding a variant fHbp according to any oneof paragraphs 0006-0010 above. The present disclosure provides an invitro host cell comprising a recombinant expression vector comprising anucleic acid encoding a variant fHbp according to any one of paragraphs0006-0010 above.

The present disclosure provides a method of eliciting an antibodyresponse in a mammal, the method comprising administering to a mammal animmunogenic composition of paragraph 0011, above. In some cases, themammal is a human. In some cases, the antibody response is abactericidal antibody response to one or more strains of N.meningitidis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts purified recombinant fHbp ID 1 mutants stained withCOOMASSIE blue on a polyacrylamide gel. Lane 1, Kaleidoscope molecularweight marker (Bio-Rad Laboratories); 2, fHbp ID 1 wild-type; 3, Q38R;4, E92K; 5, R130G; 6, S223R; 7, H248L.

FIGS. 2A and 2B depict binding of fHbp ID 1 mutants to human fH,measured by ELISA. The mean and range for replicate measurements areshown.

FIGS. 3A-3E depict binding of fHbp ID 1 mutants to human fH, measured bysurface plasmon resonance. For reference, the same data for the ID 1wild-type (WT) protein are shown in each of FIGS. 3A-3E.

FIG. 4A-4E depict binding of murine anti-fHbp monoclonal antibodies(mAb) to fHbp ID 1 mutant proteins, measured by ELISA. The mean andrange for duplicate measurements are shown.

FIGS. 5A and 5B depict bactericidal activity of serum from miceimmunized with fHbp ID 1 mutants. Each symbol represents the titer of anindividual mouse, and the horizontal bars represent the geometric meantiters. FIG. 5A depicts bactericidal activity of serum from wild-typemice immunized with fHbp ID 1 mutants. FIG. 5B depicts bactericidalactivity of serum from human fH transgenic mice immunized with fHbp ID 1mutants.

FIGS. 6A and 6B depict characterization of fHbp ID 1 single or doublemutants. FIG. 6A depicts binding of human fH to fHbp ID 1 doublemutants. FIG. 6B depicts binding of murine anti-fHbp monoclonal antibody(mAb) JAR 4 to mutants.

FIG. 7 depicts bactericidal activity of serum from wild-type miceimmunized with fHbp ID 1 single or double mutants. Each symbolrepresents the titer of an individual mouse, and the horizontal barsrepresent the geometric mean titers.

FIGS. 8A and 8B depict characterization of fHbp ID 55 mutants. FIG. 8Ashows binding of human fH to immobilized fHbp ID 55 mutants, measured byELISA and FIG. 8B shows binding of murine anti-fHbp monoclonal antibody(mAb) JAR 41 to fHbp mutant ID 55 proteins, measured by ELISA. The meanand range for two to four replicates are shown.

FIG. 9 depicts bactericidal activity of serum from wild-type miceimmunized with fHbp ID 55 mutants. Each symbol represents the titer ofan individual mouse, and the horizontal bars represent the geometricmean titers.

FIGS. 10A and 10B depict bactericidal activity of serum from miceimmunized with fHbp ID 55. FIG. 10A depicts the bactericidal activity ofserum from human fH transgenic mice immunized with the licensed Trumenbavaccine or an investigational fHbp ID 55 mutant S223R. FIG. 10B depictsthe relationship between serum human fH concentrations in individualtransgenic mice and the serum bactericidal antibody titers (circularsymbols). For comparison, the titers of wild-type (WT) mice are shown(squares).

FIGS. 11A-11D depict characterization of fHbp ID 22 mutants. FIGS.11A-11C depict binding of fHbp ID 22 mutant to human fH, measured byELISA. The mean and range of two to four replicates are shown. FIG. 11Ddepicts binding of murine anti-fHbp monoclonal antibody (mAb) JAR 4 tofHbp mutant ID 22 proteins, measured by ELISA. fHbp ID 22 wild-type (WT)and D211A mutant are shown as controls. The mean and range of duplicatemeasurements are shown.

FIGS. 12A and 12B show bactericidal activity of serum from miceimmunized with fHbp ID 22 mutants. Each symbol represents the titer ofan individual mouse, and the horizontal bars represent the geometricmean titers. FIG. 12A and FIG. 12B show bactericidal activity of serumfrom wild-type mice in two experiments to test different fHbp ID 22mutants.

FIG. 13 depicts bactericidal activity of serum from human fH transgenicmice immunized with fHbp ID 22 mutants.

FIG. 14 depicts thermal unfolding of fHbp ID 22 wild-type (WT) andL130R/G133D double mutant measured by differential scanningmicrocalorimetry.

FIG. 15A and FIG. 15B depict characterization of fHbp ID 22 triplemutants. FIG. 15A depicts binding of human fH to fHbp ID 22 triplemutants. DM refers to L130R/G133D double mutant. FIG. 15B depictsbinding of murine anti-fHbp monoclonal antibody (mAb) JAR 4 to fHbp ID22 triple mutants.

FIG. 16 depicts bactericidal activity of serum from human fH transgenicmice immunized with fHbp ID 22 triple mutants. DM refers to L130R/G133Ddouble mutant.

FIG. 17 provides a table of exemplary fHbp mutants with decreasedbinding of human fH.

FIG. 18 provides the amino acid sequence of wild-type Human factor H.

FIG. 19 provides amino acid sequences of fHbp ID 1, ID 22, and ID 55from N. meningitidis strains.

FIGS. 20-24 provide amino acid sequences of fHbp ID 1 variants.

FIGS. 25-30 provide amino acid sequences of fHbp ID 22 variants.

FIGS. 31-33 provide amino acid sequences of fHbp ID 55 variants.

FIGS. 34-36 provide amino acid sequences of fHbp ID 1 double mutantvariants.

FIGS. 37-39 provide amino acid sequences of fHbp ID 22 double and triplemutant variants.

FIG. 40 provides an amino acid sequence of NspA.

DEFINITIONS

“Factor H Binding Protein” (fHbp), which is also known in the literatureas GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and “741”refers to a class of N. meningitidis polypeptides. It is found in natureas a lipoprotein on the surface of the bacterium N. meningitidis. fHbpshave been sub-divided into three fHbp variant groups (referred to asvariant group 1 (v.1), variant group 2 (v.2), and variant group 3 (v.3)in some reports (Masignani et al. (2003) J Exp Med 197:789-99) andsub-family A and B in other reports (see, e.g., Fletcher et al. (2004)Infect Immun 72:2088-2100)) based on amino acid sequence variability andimmunologic cross-reactivity (Masignani et al. (2003) J Exp Med197:789-99). fHbp also can be classified into one of the six most commonfHbp modular groups, designated Modular Group I to Modular Group VI, asshown in Figure 2 of Vu et al. (2012) Sci. Reports 2:341. Each uniquefHbp found in N. meningitidis is also assigned an fHbp peptide IDaccording to the pubmlstorg/neisseria/fHbp/website. Because the lengthof variant 2 (v.2) fHbp protein (from strain 8047, fHbp ID 77) andvariant 3 (v.3) fHbp (from strain M1239, fHbp ID 28) differ by −1 and +7amino acid residues, respectively, from that from strain MC58 (fHbp ID1), the numbering used herein to refer to residues for v.2 and v.3 fHbpproteins differs from numbering based on the actual amino acid sequencesof these proteins. Thus, for example, reference to a leucine residue (L)at position 166 of the v.2 or v.3 fHbp sequence refers to the residue atposition 165 of the v.2 protein and at position 173 in the v.3 protein.Unless noted otherwise, the numbering of the amino acid substitutionspresent in the fHbp variants is with reference to the numbering of theamino acid residues in fHbp ID 1.

Human factor H (“human fH”) as used herein, refers to a proteincomprising an amino acid sequence as shown in FIG. 18 (SEQ ID NO:4), andnaturally-occurring human allelic variants thereof.

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from”fHbp ID 1) is meant to indicate that the polypeptide or nucleic acid hasa sequence that is based on that of a reference polypeptide or nucleicacid (e.g., a naturally occurring fHbp protein or encoding nucleicacid), and is not meant to be limiting as to the source or method inwhich the protein or nucleic acid is made. Non-limiting examples ofreference polypeptides and reference polynucleotides from which an aminoacid sequence or polynucleotide sequence may be “derived from” include anaturally-occurring fHbp, fHbp ID 1, and a non-naturally-occurring fHbp.“Derived from” in the context of bacterial strains is meant to indicatethat a strain was obtained through passage in vivo, or in in vitroculture, of a parental strain and/or is a recombinant cell obtained bymodification of a parental strain.

“Conservative amino acid substitution” refers to a substitution of oneamino acid residue for another sharing chemical and physical propertiesof the amino acid side chain (e.g., charge, size,hydrophobicity/hydrophilicity). “Conservative substitutions” areintended to include substitution within the following groups of aminoacid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr. Guidance for such substitutions can be drawnfrom alignments of amino acid sequences of polypeptides presenting theepitope of interest.

The term “protective immunity” means that a vaccine or immunizationschedule that is administered to a mammal induces an immune responsethat prevents, retards the development of, or reduces the severity of adisease that is caused by Neisseria meningitidis, or diminishes oraltogether eliminates the symptoms of the disease. Protective immunitycan be accompanied by production of bactericidal antibodies. It shouldbe noted that production of bactericidal antibodies against Neisseriameningitidis is accepted in the field as predictive of a vaccine'sprotective effect in humans. (Goldschneider et al. (1969) J. Exp. Med.129:1307; Borrow et al. (2001) Infect Immun. 69:1568).

The phrase “a disease caused by a strain of Neisseria meningitidis”encompasses any clinical symptom or combination of clinical symptomsthat are present in an infection of a human with a Neisseriameningitidis. These symptoms include but are not limited to:colonization of the upper respiratory tract (e.g. mucosa of thenasopharynx and tonsils) by a pathogenic strain of Neisseriameningitidis, penetration of the bacteria into the mucosa and thesubmucosal vascular bed, septicemia, septic shock, inflammation,hemorrhagic skin lesions, activation of fibrinolysis and of bloodcoagulation, organ dysfunction such as kidney, lung, and cardiacfailure, adrenal hemorrhaging and muscular infarction, capillaryleakage, edema, peripheral limb ischemia, respiratory distress syndrome,pericarditis and meningitis.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, in the context of an antigen (e.g., a polypeptideantigen) refers to a binding reaction that is based on and/or isprobative of the presence of the antigen in a sample which may alsoinclude a heterogeneous population of other molecules. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular antigen or antigens in a sample and do not bind in asignificant amount to other molecules present in the sample.“Specifically binds to an antibody” or “specifically immunoreactivewith” in the context of an epitope of an antigen (e.g., an epitope of apolypeptide) refers to a binding reaction which is based on and/or isprobative of the presence of the epitope in an antigen (e.g.,polypeptide) which may also include a heterogeneous population of otherepitopes, as well as a heterogeneous population of antigens. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular epitope of an antigen and do not bind in a significant amountto other epitopes present in the antigen and/or in the sample.

The phrase “in a sufficient amount to elicit an immune response” meansthat there is a detectable difference between an immune responseindicator measured before and after administration of a particularantigen preparation Immune response indicators include but are notlimited to: antibody titer or specificity, as detected by an assay suchas enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flowcytometry, immunoprecipitation, Ouchterlony immunodiffusion; bindingdetection assays of, for example, spot, Western blot or antigen arrays;cytotoxicity assays, etc.

A “surface antigen” is an antigen that is present in a surface structureof Neisseria meningitidis (e.g. the outer membrane, capsule, pili,etc.).

“Isolated” refers to an entity of interest that is in an environmentdifferent from that in which the compound may naturally occur.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified. In somecases, an isolated component (e.g., a polypeptide, such as an fHbpvariant of the present disclosure; a nucleic acid of the presentdisclosure; a recombinant vector of the present disclosure) is purified,e.g., the isolated component is at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or greater than 99%,pure.

“Enriched” means that a sample is non-naturally manipulated (e.g., by anexperimentalist or a clinician) so that a compound of interest ispresent in a greater concentration (e.g., at least a three-fold greater,at least 4-fold greater, at least 8-fold greater, at least 64-foldgreater, or more) than the concentration of the compound in the startingsample, such as a biological sample (e.g., a sample in which thecompound naturally occurs or in which it is present afteradministration), or in which the compound was made (e.g., as in abacterial polypeptide, antibody, nucleic acid, and the like).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “afactor H binding protein” includes a plurality of such factor H bindingproteins and reference to “the immunogenic composition” includesreference to one or more immunogenic compositions and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides variant factor H binding proteins (fHbp)that can elicit antibodies that are bactericidal for at least one strainof Neisseria meningitidis. The present disclosure provides compositions,including immunogenic compositions, comprising a variant fHbp of thepresent disclosure. The present disclosure provides methods of use ofvariant fHbp of the present disclosure, or a composition comprising avariant fHbp of the present disclosure.

Variant fHbp

The present disclosure provides variant fHbp that differ in amino acidsequence from a wild-type N. meningitidis fHbp by from 1 to 10 aminoacids (e.g., by from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids,from 20 amino acids to 30 amino acids, from 30 amino acids to 40 aminoacids, or from 40 amino acids to 50 amino acids, such that the variantfHbp exhibits reduced affinity to human factor H (fH), compared to areference fHbp, and where the variant fHbp elicits a bactericidal immuneresponse to one or more N. meningitidis strains when administered to amammalian host. In some cases, the variant fHbp differs in amino acidsequence from a reference wild-type N. meningitidis fHbp by no more thanfrom 1 to 10 acid substitutions. In some cases, the variant fHbp differsin amino acid sequence from a reference wild-type N. meningitidis fHbpby only one amino acid substitution.

In some cases, variant fHbp of the present disclosure comprises an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99%, amino acid sequence identity to areference fHbp sequence; where the variant fHbp comprises one or moreamino acid substitutions relative to the reference fHbp sequence suchthat the variant fHbp exhibits an affinity for human fH that is 85% orless of the binding affinity of the reference fHbp for human fH, e.g.,the variant fHbp exhibits an affinity for human fH that is from about85% to about 75%, from about 75% to about 65%, from about 65% to about55%, from about 55% to about 45%, from about 45% to about 35%, fromabout 35% to about 25%, from about 25% to about 15%, from about 15% toabout 10%, from about 10% to about 5%, from about 5% to about 2%, fromabout 2% to about 1%, or from about 1% to about 0.1%, or less than 0.1%,of the binding affinity of the affinity of the reference fHbp for humanfH; and the variant fHbp induces a bactericidal immune response to atleast one strain of N. meningitidis when administered to a mammalianhost (e.g., a human; or a non-human animal model).

A variant fHbp of the present disclosure maintains substantially thesame conformation of a reference (e.g., wild-type) fHbp that binds humanfH when the reference fHbp is in its native conformation. Whether avariant fHbp of the present disclosure maintains substantially the sameconformation of a reference (e.g., wild-type) fHbp that binds human fHcan be determined using antibodies that bind wild-type fHbp when thewild-type fHbp is in its native conformation. Such antibodies include,e.g., JAR 41; JAR 4; and JAR 31. See, e.g., Vu et al. (2012) Sci.Reports 2:341. A hybridoma producing JAR 4 monoclonal antibody has theAmerican Type Culture Collection (ATCC) number PTA-8943; see also U.S.Pat. No. 8,470,340. For example, in some cases, a variant fHbp of thepresent disclosure retains binding to JAR 4; e.g., a variant fHbp of thepresent disclosure retains at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, binding to JAR 4 of a reference fHbp (e.g.,fHbp ID 1, fHbp ID 22, or fHbp ID 55) in its native conformation.

Variants of fHbp ID 1

A “reference fHbp” from which a variant fHbp of the present disclosureis derived is in some cases fHbp ID 1. The amino acid sequence of fHbpID 1 is set out below.

fHbp ID 1: (SEQ ID NO: 1)CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNG IRHIGLAAKQ.

In some cases, a variant fHbp of the present disclosure is a variantgroup 1 fHbp. In some cases, a variant fHbp of the present disclosure isa variant group 1 fHbp, and is a modular group I fHbp. In some cases,variant fHbp of the present disclosure comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99%, amino acid sequence identity to SEQ ID NO:1; wherethe variant fHbp comprises one or more amino acid substitutions relativeto fHbp ID 1 such that the variant fHbp exhibits an affinity for humanfH that is 85% or less of the binding affinity of fHbp ID 1 for humanfH, e.g., the variant fHbp exhibits an affinity for human fH that isfrom about 85% to about 75%, from about 75% to about 65%, from about 65%to about 55%, from about 55% to about 45%, from about 45% to about 35%,from about 35% to about 25%, from about 25% to about 15%, from about 15%to about 10%, from about 10% to about 5%, from about 5% to about 2%,from about 2% to about 1%, or from about 1% to about 0.1%, or less than0.1%, of the binding affinity of the affinity of fHbp ID 1 for human fH;and the variant fHbp induces a bactericidal immune response to at leastone strain of N. meningitidis when administered to a mammalian host(e.g., a human; or a non-human animal model).

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution selected from at least one of: a)an amino acid substitution of the glutamine at amino acid 38 (Q38); b)an amino acid substitution of the glutamic acid at amino acid 92 (E92);c) a substitution of glycine for arginine at amino acid 130 (R130G); d)an amino acid substitution of the serine at amino acid 223 (S223); ande) a substitution of histidine for leucine at amino acid 248 (H248L),based on the numbering of fHbp ID 1.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the glutamine at amino acid 38(Q38). In some cases, the variant fHbp comprises a Q38R substitution.Other amino acids with positively charged or aromatic side chains, suchas lysine, histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at this position. Thus, in some cases, the variant fHbpcomprises a Q38K substitution, a Q38H substitution, a Q38F substitution,a Q38Y substitution, or a Q38W substitution. As one example, a variantfHbp of the present disclosure can comprise the amino acid sequencedepicted in FIG. 20 and set forth in SEQ ID NO:5.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the glutamic acid at amino acid92 (E92). In some cases the fHbp variant comprises an E92K substitution.Other amino acids with positively charged or aromatic side chains, suchas arginine, histidine, phenylalanine, tyrosine or tryptophan, also maybe substituted at this position. Thus, for example, in some cases thefHbp variant comprises an E92R substitution, an E92H substitution, anE92F substitution, an E92Y substitution, or an E92W substitution. As oneexample, a variant fHbp of the present disclosure can comprise the aminoacid sequence depicted in FIG. 21 and set forth in SEQ ID NO:6.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises a substitution of glycine for arginine at amino acid 130(R130G). For example, a variant fHbp of the present disclosure cancomprise the amino acid sequence depicted in FIG. 22 and set forth inSEQ ID NO:7. Other amino acids with negatively charged or aromatic sidechains, such as aspartate, glutamate, phenylalanine, tyrosine, ortryptophan, may also be substituted at R130. Thus, for example, in somecases, a variant fHbp of the present disclosure comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99%, amino acid sequence identity to SEQ IDNO:1, where the variant fHbp binds human fH with an affinity that 50% orless (e.g., from about 50% to about 45%, from about 45% to about 35%,from about 35% to about 25%, from about 25% to about 15%, from about 15%to about 10%, from about 10% to about 5%, from about 5% to about 2%,from about 2% to about 1%, or from about 1% to about 0.1%, or less than0.1%) of the affinity of fHbp ID 1 for human fH, where the variantinduces a bactericidal antibody response to at least one strain of N.meningitidis in a mammalian host, and where the variant fHbp comprisesan R130D substitution, an R130E substitution, an R130F substitution, anR130Y substitution, or an R130W substitution.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the serine at amino acid 223(S223). In some cases, the fHbp variant comprises an S223R substitution.Other amino acids with positively charged or aromatic side chains, suchas lysine, histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at this position. Thus, for example, in some cases, the fHbpvariant comprises an S223K substitution, an S223H substitution, an S223Fsubstitution, an S223Y substitution, or an S223W substitution. As oneexample, a variant fHbp of the present disclosure can comprise the aminoacid sequence depicted in FIG. 23 and set forth in SEQ ID NO:8.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises a substitution of histidine for leucine at amino acid 248(H248L). For example, a variant fHbp of the present disclosure cancomprise the amino acid sequence depicted in FIG. 24 and set forth inSEQ ID NO:9. Other amino acids with non-polar, negatively charged oraromatic side chains, such as isoleucine, valine, aspartate, glutamate,phenylalanine, tyrosine or tryptophan, also may be substituted at H248.Thus, in some cases, a variant fHbp of the present disclosure comprisesan amino acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to SEQ ID NO:1, where the variant fHbp binds human fH with anaffinity that 50% or less (e.g., from about 50% to about 45%, from about45% to about 35%, from about 35% to about 25%, from about 25% to about15%, from about 15% to about 10%, from about 10% to about 5%, from about5% to about 2%, from about 2% to about 1%, or from about 1% to about0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for human fH,where the variant induces a bactericidal antibody response to at leastone strain of N. meningitidis in a mammalian host, and where the variantfHbp comprises an H248I substitution, an H248V substitution, an H248Dsubstitution, an H248E substitution, an H248F substitution, an H248Ysubstitution, or an H248W substitution.

Combinations of Amino Acid Substitutions

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:1, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 1 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution selected from two or more of: a) anamino acid substitution of the glutamine at amino acid 38 (Q38); b) anamino acid substitution of the glutamic acid at amino acid 92 (E92); c)a substitution of glycine for arginine at amino acid 130 (R130G); d) anamino acid substitution of the serine at amino acid 223 (S223); and e) asubstitution of histidine for leucine at amino acid 248 (H248L), basedon the numbering of fHbp ID 1.

Combinations of substitutions may be included wherein the twosubstitutions are in different structural domains, and eachindependently decreases binding of fH to fHbp (e.g., one substitution inthe N-terminal domain, in combination with an amino acid substitution inthe C-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theC-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theN-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theC-terminal domain; and a second amino acid substitution within theC-terminal domain.

For example, in some cases, a variant fHbp of the present disclosurecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to SEQ ID NO:1, where the variant fHbp binds human fHwith an affinity that 50% or less (e.g., from about 50% to about 45%,from about 45% to about 35%, from about 35% to about 25%, from about 25%to about 15%, from about 15% to about 10%, from about 10% to about 5%,from about 5% to about 2%, from about 2% to about 1%, or from about 1%to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 1 for humanfH, where the variant induces a bactericidal antibody response to atleast one strain of N. meningitidis in a mammalian host, and where thevariant fHbp comprises: a) an amino acid substitution of the glutamineat amino acid 38 (Q38) and b) an amino acid substitution of the glutamicacid at amino acid 92 (E92); or where the variant fHbp comprises: a) anamino acid substitution of the glutamine at amino acid 38 (Q38) and c) asubstitution of glycine for arginine at amino acid 130 (R130G); or wherethe variant fHbp comprises a) an amino acid substitution of theglutamine at amino acid 38 (Q38) and d) an amino acid substitution ofthe serine at amino acid 223 (S223); or where the variant fHbp comprisesa) an amino acid substitution of the glutamine at amino acid 38 (Q38)and e) a substitution of histidine for leucine at amino acid 248(H248L), based on the numbering of fHbp ID 1.

As additional non-limiting examples, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:1, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 1 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: b) an amino acid substitution ofthe glutamic acid at amino acid 92 (E92) and c) a substitution ofglycine for arginine at amino acid 130 (R130G); or where the variantfHbp comprises b) an amino acid substitution of the glutamic acid atamino acid 92 (E92) and d) an amino acid substitution of the serine atamino acid 223 (S223); or where the variant fHbp comprises b) an aminoacid substitution of the glutamic acid at amino acid 92 (E92) and e) asubstitution of histidine for leucine at amino acid 248 (H248L); orwhere the variant fHbp comprises c) a substitution of glycine forarginine at amino acid 130 (R130G) and d) an amino acid substitution ofthe serine at amino acid 223 (S223); or where the variant fHbp comprisesc) a substitution of glycine for arginine at amino acid 130 (R130G) ande) a substitution of histidine for leucine at amino acid 248 (H248L); orwhere the variant fHbp comprises d) an amino acid substitution of theserine at amino acid 223 (S223) and e) a substitution of histidine forleucine at amino acid 248 (H248L), based on the numbering of fHbp ID 1.

As additional non-limiting examples, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:1, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 1 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: i) a Q38R substitution; and ii) anR130G substitution, based on the numbering of fHbp ID 1.

Also provided herein are variant fHbp proteins that include one or moresubstitutions relative to amino acid sequence of fHbp ID 1 as set forthabove and further include the substitution R41S. Exemplary variant fHbpinclude a R41S substitution and a substitution at S223, e.g., R41S/S223Rrelative to fHbp ID 1 or a R41S substitution and a H248L substitutionrelative to fHbp ID 1. In some cases, a variant fHbp of the presentdisclosure comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%,amino acid sequence identity to SEQ ID NO:1, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises two or more of the following aminoacid substitutions: a) a substitution of serine for arginine at aminoacid 41 (R41S); b) a substitution of arginine for serine at amino acid223 (S223R); c) a substitution of leucine for histidine at amino acid248 (H248L), based on the numbering of fHbp ID 1.

Also disclosed herein are variant fHbp proteins that include one or moresubstitutions relative to amino acid sequence of fHbp ID 1 as set forthabove and further include the substitutions disclosed in US2011/0256180,which is herein incorporated by reference in its entirety.

Variants of fHbp ID 22

A “reference fHbp” from which a variant fHbp of the present disclosureis derived is in some cases fHbp ID 22. The amino acid sequence of fHbpID 22 is set out below.

fHbp ID 22: (SEQ ID NO: 2)CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKV HEIGIAGKQ.

In some cases, a variant fHbp of the present disclosure is a variantgroup 2 fHbp. In some cases, a variant fHbp of the present disclosure isa variant group 2 fHbp, and is a modular group III fHbp. In some cases,variant fHbp of the present disclosure comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99%, amino acid sequence identity to SEQ ID NO:2; wherethe variant fHbp comprises one or more amino acid substitutions relativeto fHbp ID 22 such that the variant fHbp exhibits an affinity for humanfH that is 85% or less of the binding affinity of fHbp ID 22 for humanfH, e.g., the variant fHbp exhibits an affinity for human fH that isfrom about 85% to about 75%, from about 75% to about 65%, from about 65%to about 55%, from about 55% to about 45%, from about 45% to about 35%,from about 35% to about 25%, from about 25% to about 15%, from about 15%to about 10%, from about 10% to about 5%, from about 5% to about 2%,from about 2% to about 1%, or from about 1% to about 0.1%, or less than0.1%, of the binding affinity of the affinity of fHbp ID 22 for humanfH; and the variant fHbp induces a bactericidal immune response to atleast one strain of N. meningitidis when administered to a mammalianhost (e.g., a human; or a non-human animal model).

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution selected from at least one of: a) asubstitution of isoleucine for asparagine at amino acid 115 (N115I); b)a substitution of glycine for aspartic acid at amino acid 121 (D121G);c) a substitution of threonine for serine at amino acid 128 (S128T); d)an amino acid substitution of the valine at position 131 (V131); e) anamino acid substitution of the lysine at position 219 (K219); f) anamino acid substitution of the glycine at position 220 (G220), relativeto the amino acid sequence of fHbp ID 22. As noted herein, the numberingof the amino acid residue is based on that of fHbp ID 1.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises a substitution of isoleucine for asparagine at amino acid 115(N115I). For example, a variant fHbp of the present disclosure cancomprise the amino acid sequence depicted in FIG. 25 and set forth inSEQ ID NO:10. Other amino acids with non-polar, positively charged oraromatic side chains, such as valine, leucine, lysine, arginine,histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at N115. Thus, for example, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises an N115V substitution, an N115Lsubstitution, an N115K substitution, an N115R substitution, an N115Hsubstitution, an N115F substitution, an N115Y substitution, or an N115Wsubstitution relative to amino acid sequence of fHbp ID 22.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises a substitution of glycine for aspartic acid at amino acid 121(D121G). For example, a variant fHbp of the present disclosure cancomprise the amino acid sequence depicted in FIG. 26 and set forth inSEQ ID NO:11. Other amino acids with non-polar, positively charged oraromatic side chains, such as leucine, isoleucine, valine, lysine,arginine, histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at D121. Thus, for example, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises a D121L substitution, a D121Isubstitution, a D121V substitution, a D121K substitution, a D121Rsubstitution, a D121H substitution, a D121F substitution, a D121Ysubstitution, or a D121W substitution relative to amino acid sequence offHbp ID 22.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises a substitution of threonine for serine at amino acid 128(S128T). For example, a variant fHbp of the present disclosure cancomprise the amino acid sequence depicted in FIG. 27 and set forth inSEQ ID NO:12. Other amino acids with polar, charged or aromatic sidechains, such as methionine, asparagine, glutamine, aspartate, glutamate,lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, alsomay be substituted at 5128. Thus, for example, in some cases, a variantfHbp of the present disclosure comprises an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to SEQ ID NO:2, where thevariant fHbp binds human fH with an affinity that 50% or less (e.g.,from about 50% to about 45%, from about 45% to about 35%, from about 35%to about 25%, from about 25% to about 15%, from about 15% to about 10%,from about 10% to about 5%, from about 5% to about 2%, from about 2% toabout 1%, or from about 1% to about 0.1%, or less than 0.1%) of theaffinity of fHbp ID 22 for human fH, where the variant induces abactericidal antibody response to at least one strain of N. meningitidisin a mammalian host, and where the variant fHbp comprises an S128Msubstitution, an S128N substitution, an S128D substitution, an S128Esubstitution, an S128K substitution, an S128R substitution, an S128Hsubstitution, an S128F substitution, an S128Y substitution, or an S128Wsubstitution relative to amino acid sequence of fHbp ID 22.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the valine at position 131(V131). In some cases, the fHbp variant comprises a V131D substitution.Other amino acids with charged or aromatic side chains, such asglutamate, lysine, arginine, histidine, phenylalanine, tyrosine ortryptophan, also may be substituted at this position. Thus, for example,in some cases, the fHbp variant comprises a V131E substitution, a V131Ksubstitution, a V131R substitution, a V131H substitution, a V131Fsubstitution, a V131Y substitution, or a V131W substitution. As oneexample, a variant fHbp of the present disclosure can comprise the aminoacid sequence depicted in FIG. 28 and set forth in SEQ ID NO:13 relativeto amino acid sequence of fHbp ID 22.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the lysine at position 219(K219). In some cases, the fHbp variant comprises a K219N substitution.Other amino acids with polar, negatively charged or aromatic sidechains, such as glutamine, aspartate, glutamate, phenylalanine, tyrosineor tryptophan, also may be substituted at this position. Thus, forexample, in some cases, the fHbp variant comprises a K219Q substitution,a K219D substitution, a K219E substitution, a K219F substitution, aK219Y substitution, or a K219W substitution. As one example, a variantfHbp of the present disclosure can comprise the amino acid sequencedepicted in FIG. 29 and set forth in SEQ ID NO:14.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the glycine at position 220(G220). In some cases, the fHbp variant comprises a G220S substitution.Other amino acids with polar, charged or aromatic side chains, such asasparagine, glutamine, aspartate, glutamate, lysine, arginine,histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at this position. Thus, for example, in some cases, the fHbpvariant comprises a G220N substitution, a G220Q substitution, a G220Dsubstitution, a G220E substitution, a G220K substitution, a G220Rsubstitution, a G220H substitution, a G220F substitution, a G220Ysubstitution, or a G220W substitution. For example, a variant fHbp ofthe present disclosure can comprise the amino acid sequence depicted inFIG. 30 and set forth in SEQ ID NO:15.

Combinations of Amino Acid Substitutions

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:2, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 22 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution selected from two or more of: a) asubstitution of isoleucine for asparagine at amino acid 115 (N115I); b)a substitution of glycine for aspartic acid at amino acid 121 (D121G);c) a substitution of threonine for serine at amino acid 128 (S128T); d)an amino acid substitution of the valine at position 131 (V131); e) anamino acid substitution of the lysine at position 219 (K219); f) anamino acid substitution of the glycine at position 220 (G220), relativeto amino acid sequence of fHbp ID 22. As noted herein, the numbering ofthe residues is based on the numbering of the amino acids in fHbp ID 1.

Combinations of substitutions may be included wherein the twosubstitutions are in different structural domains, and eachindependently decreases binding of fH to fHbp (e.g., one substitution inthe N-terminal domain, in combination with an amino acid substitution inthe C-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theC-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theN-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theC-terminal domain; and a second amino acid substitution within theC-terminal domain.

For example, in some cases, a variant fHbp of the present disclosurecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to SEQ ID NO:2, where the variant fHbp binds human fHwith an affinity that 50% or less (e.g., from about 50% to about 45%,from about 45% to about 35%, from about 35% to about 25%, from about 25%to about 15%, from about 15% to about 10%, from about 10% to about 5%,from about 5% to about 2%, from about 2% to about 1%, or from about 1%to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 forhuman fH, where the variant induces a bactericidal antibody response toat least one strain of N. meningitidis in a mammalian host, and wherethe variant fHbp comprises: a) a substitution of isoleucine forasparagine at amino acid 115 (N115I) and b) a substitution of glycinefor aspartic acid at amino acid 121 (D121G); or where the variant fHbpcomprises: a) a substitution of isoleucine for asparagine at amino acid115 (N115I) and c) a substitution of threonine for serine at amino acid128 (S128T); or where the variant fHbp comprises: a) a substitution ofisoleucine for asparagine at amino acid 115 (N115I) and d) an amino acidsubstitution of the valine at position 131 (V131); or where the variantfHbp comprises: a) a substitution of isoleucine for asparagine at aminoacid 115 (N115I) and e) an amino acid substitution of the lysine atposition 219 (K219); or where the variant fHbp comprises: a) asubstitution of isoleucine for asparagine at amino acid 115 (N115I) andf) an amino acid substitution of the glycine at position 220 (G220),relative to amino acid sequence of fHbp ID 22, the numbering of thesubstituted residue based on the numbering of amino acid sequence offHbp ID 1.

As additional non-limiting examples, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: b) a substitution of glycine foraspartic acid at amino acid 121 (D121G) and c) a substitution ofthreonine for serine at amino acid 128 (S128T); or where the variantfHbp comprises: b) a substitution of glycine for aspartic acid at aminoacid 121 (D121G) and d) an amino acid substitution of the valine atposition 131 (V131); or where the variant fHbp comprises: b) asubstitution of glycine for aspartic acid at amino acid 121 (D121G) ande) an amino acid substitution of the lysine at position 219 (K219); orwhere the variant fHbp comprises: b) a substitution of glycine foraspartic acid at amino acid 121 (D121G) and f) an amino acidsubstitution of the glycine at position 220 (G220), relative to aminoacid sequence of fHbp ID 22. The numbering of the substituted residue(s)is based on the numbering of amino acid sequence of fHbp ID 1.

As additional non-limiting examples, in some cases, a variant fHbp ofthe present disclosure comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: c) a substitution of threonine forserine at amino acid 128 (S128T) and d) an amino acid substitution ofthe valine at position 131 (V131); or where the variant fHbp comprises:c) a substitution of threonine for serine at amino acid 128 (S128T) ande) an amino acid substitution of the lysine at position 219 (K219); orwhere the variant fHbp comprises: c) a substitution of threonine forserine at amino acid 128 (S128T) and f) an amino acid substitution ofthe glycine at position 220 (G220); or where the variant fHbp comprises:d) an amino acid substitution of the valine at position 131 (V131) ande) an amino acid substitution of the lysine at position 219 (K219); orwhere the variant fHbp comprises: d) an amino acid substitution of thevaline at position 131 (V131) and f) an amino acid substitution of theglycine at position 220 (G220); or where the variant fHbp comprises: e)an amino acid substitution of the lysine at position 219 (K219) and f)an amino acid substitution of the glycine at position 220 (G220),),relative to amino acid sequence of fHbp ID 22. The numbering of thesubstituted residue(s) is based on the numbering of amino acid sequenceof fHbp ID 1.

Combinations of substitutions may be included wherein the twosubstitutions are in different structural domains, and eachindependently decreases binding of fH to fHbp (e.g. one from theN-terminal domain (e.g., N115I, D121G, S128T or V131D) in combinationwith one from the C-terminal domain (e.g., D211A, K219N, G220S).

For example, in some cases, a variant fHbp of the present disclosurecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to SEQ ID NO:2, where the variant fHbp binds human fHwith an affinity that 50% or less (e.g., from about 50% to about 45%,from about 45% to about 35%, from about 35% to about 25%, from about 25%to about 15%, from about 15% to about 10%, from about 10% to about 5%,from about 5% to about 2%, from about 2% to about 1%, or from about 1%to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 22 forhuman fH, where the variant induces a bactericidal antibody response toat least one strain of N. meningitidis in a mammalian host, and wherethe variant fHbp comprises: i) an N115I substitution; and ii) a D211Asubstitution.

As another example, in some cases, a variant fHbp of the presentdisclosure comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%,amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: i) an N115I substitution; and ii)a K219N substitution.

As another example, in some cases, a variant fHbp of the presentdisclosure comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%,amino acid sequence identity to SEQ ID NO:2, where the variant fHbpbinds human fH with an affinity that 50% or less (e.g., from about 50%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%) of the affinity of fHbpID 22 for human fH, where the variant induces a bactericidal antibodyresponse to at least one strain of N. meningitidis in a mammalian host,and where the variant fHbp comprises: i) an N115I substitution; and ii)a G220S substitution.

Also disclosed herein are variant fHbp polypeptides that have increasedthermal stability compared to wild type fHbp ID22. In some cases, thevariant fHbp may include the substitutions L130R and G133D relative tofHbp ID 22 (SEQ ID NO:2), wherein the variant fHbp comprises an aminoacid sequence having greater than 85% amino acid sequence identity toSEQ ID NO:2, wherein the variant fHbp binds human factor H (fH) with anaffinity that is 50% or less of the affinity of fHbp ID 22 for human fH,wherein the variant induces a bactericidal antibody response in amammalian host, and the variant has a higher thermal stability that WTfHbp ID 22. The thermal stability of the variant fHbp may be higher thana WT fHbp (e.g., fHbp ID 22) by at least 5° C., 10° C., 15° C., 20° C.,or more, e.g., higher by 5° C.-30° C., 5° C.-25° C., 5° C.-20° C., 10°C.-20° C., or 15° C.-20° C. As used herein, “thermal stability” refersto stability of a protein when exposed to higher temperature; a thermalstability variant protein maintains its conformation at a highertemperature than a wild type protein. For example, the variant fHbp,that include the double mutation that increases thermal stabilitycompared to thermal stability of wild type (WT) fHbp, e.g., WT fHbp ID22, may unfold at a higher temperature compared to WT fHbp. In certaincases, the N-terminal domain of the variant fHbp may unfold at a highertemperature than the N-terminal domain of the WT fHbp (e.g., fHbp ID22).

In certain embodiments, a variant of factor H binding protein (fHbp) isdisclosed, wherein the variant comprises amino acid substitutions L130Rand G133D and at least one of the substitutions: R80A, N115I, D121G,S128T, V131, D211A, E218A, K219 (e.g., K219N), G220 (e.g., G220S),E248A, G236I, T221A, and H223A relative to fHbp ID 22 (SEQ ID NO:2),wherein the variant comprises an amino acid sequence having greater than85% amino acid sequence identity to SEQ ID NO:2, wherein the variantfHbp binds human factor H (fH) with an affinity that is 50% or less ofthe affinity of fHbp ID 22 for human fH, and wherein the variant inducesa bactericidal antibody response in a mammalian host.

In some cases, the variant fHbp may include a combination ofsubstitutions, such as, L130R, G133D, and at least one amino acidsubstitution selected from: a) N115I; b) D121G; c) S128T; d) V131D; e)K219 (e.g., K219N); and f) G220 (e.g., G220S), wherein the amino acidsubstitutions are relative to fHbp ID 22 (SEQ ID NO:2), wherein thevariant fHbp comprises an amino acid sequence having greater than 85%amino acid sequence identity to SEQ ID NO:2, wherein the variant fHbpbinds human factor H (fH) with an affinity that is 50% or less of theaffinity of fHbp ID 22 for human fH, and wherein the variant induces abactericidal antibody response in a mammalian host.

In certain embodiments, a variant of factor H binding protein (fHbp) isdisclosed, wherein the variant comprises amino acid substitutions L130Rand G133D and at least one of the substitutions: R80A, D211A, E218A,E248A, G236I, T221A, and H223A relative to fHbp ID 22 (SEQ ID NO:2),wherein the variant comprises an amino acid sequence having greater than85% amino acid sequence identity to SEQ ID NO:2, wherein the variantfHbp binds human factor H (fH) with an affinity that is 50% or less ofthe affinity of fHbp ID 22 for human fH, and wherein the variant inducesa bactericidal antibody response in a mammalian host.

Exemplary variant fHbp include a polypeptide having an amino acidsequence having greater than 85% amino acid sequence identity (e.g., atleast 90% identity, at least 95%, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity to SEQ ID NO:2,and including the following substitutions relative to the amino acidsequence of SEQ ID NO:2: L130R and G133D; L130R, G133D, and K219N; orL130R, G133D, and G220S.

Also disclosed herein are variant fHbp proteins that include one or moresubstitutions relative to amino acid sequence of fHbp ID 22 as set forthabove and further include the substitutions disclosed in US2011/0256180,which is herein incorporated by reference in its entirety.

Variants of fHbp ID 55

A “reference fHbp” from which a variant fHbp of the present disclosureis derived is in some cases fHbp ID 55. The amino acid sequence of fHbpID 55 is set out below.

fHbp ID 55: (SEQ ID NO: 3)CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEV ETANGIHHIGLAAKQ.

In some cases, a variant fHbp of the present disclosure is a variantgroup 1 fHbp. In some cases, a variant fHbp of the present disclosure isa variant group 1 fHbp, and is a modular group IV fHbp.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3; where the variant fHbp comprises one or more amino acidsubstitutions relative to fHbp ID 55 such that the variant fHbp exhibitsan affinity for human fH that is 85% or less of the binding affinity offHbp ID 55 for human fH, e.g., the variant fHbp exhibits an affinity forhuman fH that is from about 85% to about 75%, from about 75% to about65%, from about 65% to about 55%, from about 55% to about 45%, fromabout 45% to about 35%, from about 35% to about 25%, from about 25% toabout 15%, from about 15% to about 10%, from about 10% to about 5%, fromabout 5% to about 2%, from about 2% to about 1%, or from about 1% toabout 0.1%, or less than 0.1%, of the binding affinity of the affinityof fHbp ID 55 for human fH; and the variant fHbp induces a bactericidalimmune response to at least one strain of N. meningitidis whenadministered to a mammalian host (e.g., a human; or a non-human animalmodel).

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 55 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution selected from at least one of: a)an amino acid substitution of the glutamic acid at position 92 (E92); b)an amino acid substitution of the serine at position 223 (S223); and c)an amino acid substitution of the histidine at position 248 (H248),relative to the amino acid sequence of fHbp ID 55, where the numberingof the amino acid residues is based on the numbering of fHbp ID 1.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 55 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the glutamic acid at position 92(E92). In some cases, the fHbp variant comprises an E92K substitution.Other amino acids with positively charged or aromatic side chains, suchas arginine, histidine, phenylalanine, tyrosine or tryptophan, also maybe substituted at this position. Thus, for example, in some cases, thefHbp variant comprises an E92R substitution, an E92H substitution, anE92F substitution, an E92Y substitution, or an E92W substitution. As oneexample, a variant fHbp of the present disclosure can comprise the aminoacid sequence depicted in FIG. 31 and set forth in SEQ ID NO:16.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 55 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the serine at position 223(S223). In some cases, the fHbp variant comprises an S223R substitution.Other amino acids with positively charged or aromatic side chains, suchas lysine, histidine, phenylalanine, tyrosine or tryptophan, also may besubstituted at this position. Thus, for example, in some cases, the fHbpvariant comprises an S223K substitution, an S223H substitution, an S223Fsubstitution, an S223Y substitution, or an S223W substitution. As oneexample, a variant fHbp of the present disclosure can comprise the aminoacid sequence depicted in FIG. 32 and set forth in SEQ ID NO:17.

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 55 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises an amino acid substitution of the histidine at position 248(H248). In some cases, the fHbp variant comprises an H248L substitution.Other amino acids with non-polar, negatively charged or aromatic sidechains, such as isoleucine, valine, aspartate, glutamate, phenylalanine,tyrosine or tryptophan, also may be substituted at this position. Thus,for example, in some cases, the fHbp variant comprises an H248Isubstitution, an H248V substitution, an H248D substitution, an H248Esubstitution, an H248F substitution, an H248Y substitution, or an H248Wsubstitution. As one example, a variant fHbp of the present disclosurecan comprise the amino acid sequence depicted in FIG. 33 and set forthin SEQ ID NO:18.

Combinations of Amino Acid Substitutions

In some cases, a variant fHbp of the present disclosure comprises anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto SEQ ID NO:3, where the variant fHbp binds human fH with an affinitythat 50% or less (e.g., from about 50% to about 45%, from about 45% toabout 35%, from about 35% to about 25%, from about 25% to about 15%,from about 15% to about 10%, from about 10% to about 5%, from about 5%to about 2%, from about 2% to about 1%, or from about 1% to about 0.1%,or less than 0.1%) of the affinity of fHbp ID 55 for human fH, where thevariant induces a bactericidal antibody response to at least one strainof N. meningitidis in a mammalian host, and where the variant fHbpcomprises two or more amino acid substitutions selected from the groupconsisting of: a) an amino acid substitution of the glutamic acid atposition 92 (E92); b) an amino acid substitution of the serine atposition 223 (S223); and c) an amino acid substitution of the histidineat position 248 (H248), relative to fHbp ID 55, where the numbering ofthe residue is based on the numbering of amino acids in the sequence forfHbp ID 1.

Combinations of substitutions may be included wherein the twosubstitutions are in different structural domains, and eachindependently decreases binding of fH to fHbp (e.g., one substitution inthe N-terminal domain, in combination with an amino acid substitution inthe C-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theC-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theN-terminal domain; and a second amino acid substitution within theN-terminal domain. In some cases, a variant fHbp of the presentdisclosure comprises a first amino acid substitution within theC-terminal domain; and a second amino acid substitution within theC-terminal domain.

For example, in some cases, a variant fHbp of the present disclosurecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to SEQ ID NO:3, where the variant fHbp binds human fHwith an affinity that 50% or less (e.g., from about 50% to about 45%,from about 45% to about 35%, from about 35% to about 25%, from about 25%to about 15%, from about 15% to about 10%, from about 10% to about 5%,from about 5% to about 2%, from about 2% to about 1%, or from about 1%to about 0.1%, or less than 0.1%) of the affinity of fHbp ID 55 forhuman fH, where the variant induces a bactericidal antibody response toat least one strain of N. meningitidis in a mammalian host, and wherethe variant fHbp comprises: a) an amino acid substitution of theglutamic acid at position 92 (E92) and b) an amino acid substitution ofthe serine at position 223 (S223); or where the variant fHbp comprises:a) an amino acid substitution of the glutamic acid at position 92 (E92)and c) an amino acid substitution of the histidine at position 248(H248); or where the variant fHbp comprises: b) an amino acidsubstitution of the serine at position 223 (S223) and c) an amino acidsubstitution of the histidine at position 248 (H248); or where thevariant fHbp comprises: a) an amino acid substitution of the glutamicacid at position 92 (E92) and b) an amino acid substitution of theserine at position 223 (S223) and c) an amino acid substitution of thehistidine at position 248 (H248), relative to fHbp ID 55, where thenumbering of the residue is based on the numbering of amino acids in thesequence for fHbp ID 1.

Also disclosed herein are variant fHbp proteins that include one or moresubstitutions relative to amino acid sequence of fHbp ID 55 as set forthabove and further include the substitutions disclosed in US2011/0256180,which is herein incorporated by reference in its entirety.

Fusion Polypeptides

A variant fHbp of the present disclosure can be a fusion polypeptide,e.g., a polypeptide comprising a variant fHbp as described above, and aheterologous polypeptide (e.g., a fusion partner). The fusion partnercan be at the N-terminus of the variant fHbp, at the C-terminus of thevariant fHbp, or at an internal site within the fHbp.

Suitable fusion partners include peptides and polypeptides that conferenhanced stability in vivo (e.g., enhanced serum half-life); provideease of purification, e.g., (His)_(n), e.g., 6His, and the like; providefor secretion of the fusion protein from a cell; provide an epitope tag,e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:26), FLAG(e.g., DYKDDDDK; SEQ ID NO:27), c-myc (e.g., EQKLISEEDL; SEQ ID NO:28),and the like; provide a detectable signal, e.g., an enzyme thatgenerates a detectable product (e.g., β-galactosidase, luciferase), or aprotein that is itself detectable, e.g., a green fluorescent protein, ayellow fluorescent protein, etc.; provides for multimerization, e.g., amultimerization domain such as an Fc portion of an immunoglobulin; andthe like.

Methods of Production

The fHbps of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis). Where the subject fHbp is produced usingrecombinant techniques, the methods can involve any suitable constructand any suitable host cell, which can be a prokaryotic or eukaryoticcell, usually a bacterial or yeast host cell, more usually a bacterialcell. Methods for introduction of genetic material into host cellsinclude, for example, transformation, electroporation, conjugation,calcium phosphate methods and the like. The method for transfer can beselected so as to provide for stable expression of the introducedfHbp-encoding nucleic acid. The fHbp-encoding nucleic acid can beprovided as an inheritable episomal element (e.g., plasmid) or can begenomically integrated.

The present disclosure provides nucleic acids (including isolatednucleic acids) that comprise a nucleotide sequence encoding a fHbpvariant of the present disclosure. In some embodiments, the nucleotidesequence encoding the fHbp variant is operably linked to atranscriptional control element, e.g., a promoter. The promoter is insome cases constitutive. The promoter is in some cases inducible. Insome cases, the promoter is suitable for use (e.g., active in) aprokaryotic host cell. In some cases, the promoter is suitable for use(e.g., active in) a eukaryotic host cell.

In some instances, a nucleic acid comprising a nucleotide sequenceencoding a fHbp variant of the present disclosure is present in anexpression vector. The present disclosure provides a recombinantexpression vector (e.g., an isolated recombinant expression vector) thatcomprises a nucleotide sequence encoding a fHbp variant of the presentdisclosure. In some embodiments, the nucleotide sequence encoding thefHbp variant is operably linked to a transcriptional control element,e.g., a promoter. The promoter is in some cases constitutive. Thepromoter is in some cases inducible. In some cases, the promoter issuitable for use (e.g., active in) a prokaryotic host cell. In somecases, the promoter is suitable for use (e.g., active in) a eukaryotichost cell.

Suitable vectors for transferring fHbp-encoding nucleic acid can vary incomposition. Integrative vectors can be conditionally replicative orsuicide plasmids, bacteriophages, and the like. The constructs caninclude various elements, including for example, promoters, selectablegenetic markers (e.g., genes conferring resistance to antibiotics (forinstance kanamycin, erythromycin, chloramphenicol, or gentamycin)),origin of replication (to promote replication in a host cell, e.g., abacterial host cell), and the like. The choice of vector will dependupon a variety of factors such as the type of cell in which propagationis desired and the purpose of propagation. Certain vectors are usefulfor amplifying and making large amounts of the desired DNA sequence.Other vectors are suitable for expression in cells in culture. Stillother vectors are suitable for transfer and expression in cells in awhole animal. The choice of appropriate vector is well within the skillof the art. Many such vectors are available commercially.

In one example, the vector is an expression vector based on episomalplasmids containing selectable drug resistance markers and elements thatprovide for autonomous replication in different host cells (e.g., inboth E. coli and N. meningitidis). One example of such a “shuttlevector” is the plasmid pFP10 (Pagotto et al. (2000) Gene 244:13-19).

Constructs (recombinant vectors) can be prepared by, for example,inserting a polynucleotide of interest into a construct backbone,typically by means of DNA ligase attachment to a cleaved restrictionenzyme site in the vector. Alternatively, the desired nucleotidesequence can be inserted by homologous recombination or site-specificrecombination. Typically homologous recombination is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence, while site-specific recombination can beaccomplished through use of sequences that facilitate site-specificrecombination (e.g., cre-lox, att sites, etc.). Nucleic acid containingsuch sequences can be added by, for example, ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding the subject fHbp, may provide forpropagating the subject nucleic acids, or both.

Examples of vectors that may be used include but are not limited tothose derived from recombinant bacteriophage DNA, plasmid DNA or cosmidDNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118,119 and the M13 mp series of vectors may be used. pET21 is also anexpression vector that may be used. Bacteriophage vectors may includeλgt10, λgt11, λgt18-23, λZAP/R and the EMBL series of bacteriophagevectors. Further vectors that may be utilized include, but are notlimited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274,COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

For expression of a subject fHbp, an expression cassette may beemployed. Thus, the present disclosure provides a recombinant expressionvector comprising a subject nucleic acid. The expression vector providestranscriptional and translational regulatory sequences, and may providefor inducible or constitutive expression, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and a transcriptional and translational terminationregion. These control regions may be native to an fHbp from which thesubject fHbp is derived, or may be derived from exogenous sources. Ingeneral, the transcriptional and translational regulatory sequences mayinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. Promoters can beeither constitutive or inducible, and can be a strong constitutivepromoter (e.g., T7, and the like).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host may be present to facilitate selection of cellscontaining the vector. In addition, the expression construct may includeadditional elements. For example, the expression vector may have one ortwo replication systems, thus allowing it to be maintained in organisms,for example in mammalian or insect cells for expression and in aprokaryotic host for cloning and amplification. In addition theexpression construct may contain a selectable marker gene to allow theselection of transformed host cells. Selection genes are well known inthe art and will vary with the host cell used.

It should be noted that fHbps of the present disclosure may compriseadditional elements, such as a detectable label, e.g., a radioactivelabel, a fluorescent label, a biotin label, an immunologicallydetectable label (e.g., a hemagglutinin tag, a poly-Histidine tag) andthe like. Additional elements of fHbp can be provided to facilitateisolation (e.g., biotin tag, immunologically detectable tag) throughvarious methods (e.g., affinity capture, etc.). The subject fHbp canoptionally be immobilized on a support through covalent or non-covalentattachment.

Isolation and purification of fHbp can be accomplished according tomethods known in the art. For example, fHbp can be isolated from alysate of cells genetically modified to express a fHbp, or from asynthetic reaction mix, by immunoaffinity purification, which generallyinvolves contacting the sample with an anti-fHbp antibody (e.g., ananti-fHbp monoclonal antibody (mAb), such as a JAR 4 MAb or otherappropriate JAR MAb known in the art), washing to removenon-specifically bound material, and eluting specifically bound fHbp.Isolated fHbp can be further purified by dialysis and other methodsnormally employed in protein purification methods. In one example, thefHbp can be isolated using metal chelate chromatography methods.

Host Cells

Any of a number of suitable host cells can be used in the production offHbp. In general, the fHbp described herein may be expressed inprokaryotes or eukaryotes, e.g., bacteria such as Escherichia coli orNeisseria (e.g., N. meningitidis) in accordance with conventionaltechniques. Thus, the present disclosure further provides a geneticallymodified in vitro host cell, which contains a nucleic acid encoding asubject fHbp. Host cells for production (including large scaleproduction) of a subject fHbp can be selected from any of a variety ofavailable host cells. Examples of host cells for expression includethose of a prokaryotic or eukaryotic unicellular organism, such asbacteria (e.g., Escherichia coli strains), yeast (e.g., Saccharomycescerevisiae, Pichia spp., and the like), and may include host cellsoriginally derived from a higher organism such as insects, vertebrates,e.g., mammals. Suitable mammalian cell lines include, but are notlimited to, HeLa cells (e.g., American Type Culture Collection (ATCC)No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCCNo. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells(ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK)cells (ATCC No. CRL1573), HLHepG2 cells, and the like.). In some cases,bacterial host cells and yeast host cells are of particular interest forsubject fHbp production.

Subject fHbps can be prepared in substantially pure or substantiallyisolated form (i.e., substantially free from other Neisserial or hostcell polypeptides) or substantially isolated form. The subject fHbp canbe present in a composition that is enriched for the polypeptiderelative to other components that may be present (e.g., otherpolypeptides or other host cell components). Purified subject fHbp canbe provided such that the polypeptide is present in a composition thatis substantially free of other expressed polypeptides, e.g., less than90%, usually less than 60% and more usually less than 50% of thecomposition is made up of other expressed polypeptides.

Host Cells for Vesicle Production

Where a subject fHbp is to be provided in a membrane vesicle (asdiscussed in more detail below), a Neisserial host cell is geneticallymodified to express a subject fHbp. Any of a variety of Neisseria spp.strains can be modified to produce a subject fHbp, and, optionally,which produce or can be modified to produce other antigens of interest,such as PorA, can be used in the methods disclosed herein.

Methods and vectors to provide for genetic modification of Neisserialstrains and expression of a desired polypeptide are known in the art.Examples of vectors and methods can be found in WO 02/09746 and O'Dwyeret al. (2004) Infect Immun 72:6511-80. Strong promoters, particularlyconstitutive strong promoters are of particular interest. Examples ofpromoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB,lgtF, opa, p110, lst, hpuAB, and rmp.

Pathogenic Neisseria spp. or strains derived from pathogenic Neisseriaspp., particularly strains pathogenic for humans or derived from strainspathogenic or commensal for humans, are of particular interest for usein membrane vesicle production. Examples of Neisserial spp. include N.meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N.polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N.elongata, and the like.

N. meningitidis strains are of particular interest for geneticmodification to express the subject fHbps and for use in vesicleproduction. The strain used for vesicle production can be selectedaccording to a number of different characteristics that may be desired.For example, the strain may be selected according to: a desired PorAtype (a “serosubtype”), capsular group, serotype, and the like;decreased capsular polysaccharide production; and the like. For example,the production strain can produce any desired PorA polypeptide, and mayexpress one or more PorA polypeptides (either naturally or due togenetic engineering). Examples of strains include those that produce aPorA polypeptide which confers a serosubtype of P1.7,16; P1.19,15;P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4; P1.12,13; as well asvariants of such PorA polypeptides which may or may not retainreactivity with conventional serologic reagents used in serosubtyping.Also of interest are PorA polypeptides characterized according to PorAvariable region (VR) typing (see, e.g., Russell et al. (2004) EmergingInfect Dis 10:674-678; Sacchi C T et al. (1998) Clin Diagn Lab Immunol5:845-55; Sacchi et al (2000) J. Infect Dis 182:1169-1176). Asubstantial number of distinct VR types have been identified, which canbe classified into VR1 and VR2 family “prototypes”. A web-accessibledatabase describing this nomenclature and its relationship to previoustyping schemes is found at neisseria.org/nm/typing/pora. Alignments ofcertain PorA VR1 and VR2 types are provided in Russell et al. (2004)Emerging Infect Dis 10:674-678.

Alternatively or in addition, the production strain can be a capsuledeficient strain. Capsule deficient strains can provide vesicle-basedvaccines that provide for a reduced risk of eliciting a significantautoantibody response in a subject to whom the vaccine is administered(e.g., due to production of antibodies that cross-react with sialic acidon host cell surfaces). “Capsule deficient” or “deficient in capsularpolysaccharide” as used herein refers to a level of capsularpolysaccharide on the bacterial surface that is lower than that of anaturally-occurring strain or, where the strain is genetically modified,is lower than that of a parental strain from which the capsule deficientstrain is derived. A capsule deficient strain includes strains that aredecreased in surface capsular polysaccharide production by at least 10%,20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includesstrains in which capsular polysaccharide is not detectable on thebacterial surface (e.g., by whole cell enzyme-linked immunosorbent assay(ELISA) using an anti-capsular polysaccharide antibody).

Capsule deficient strains include those that are capsule deficient dueto a naturally-occurring or recombinantly-generated geneticmodification. Naturally-occurring capsule deficient strains (see, e.g.,Dolan-Livengood et al. (2003) J. Infect. Dis. 187:1616-28), as well asmethods of identifying and/or generating capsule-deficient strains (see,e.g., Fisseha et al. (2005) Infect. Immun. 73:4070-4080; Stephens et al.(1991) Infect Immun 59:4097-102; Frosch et al. (1990) Mol Microbiol.4:1215-1218) are known in the art.

Modification of a Neisserial host cell to provide for decreasedproduction of capsular polysaccharide may include modification of one ormore genes involved in capsule synthesis, where the modificationprovides for, for example, decreased levels of capsular polysacchariderelative to a parent cell prior to modification. Such geneticmodifications can include changes in nucleotide and/or amino acidsequences in one or more capsule biosynthesis genes rendering the straincapsule deficient (e.g., due to one or more insertions, deletions,substitutions, and the like in one or more capsule biosynthesis genes).Capsule deficient strains can lack or be non-functional for one or morecapsule genes.

Of particular interest are strains that are deficient in sialic acidbiosynthesis. Such strains can provide for production of vesicles thathave reduced risk of eliciting anti-sialic acid antibodies thatcross-react with human sialic acid antigens, and can further provide forimproved manufacturing safety. Strains having a defect in sialic acidbiosynthesis (due to either a naturally occurring modification or anengineered modification) can be defective in any of a number ofdifferent genes in the sialic acid biosynthetic pathway. Of particularinterest are strains that are defective in a gene product encoded by theN-acetylglucosamine-6-phosphate 2-epimerase gene (known as synXAAF40537.1 or siaA AAA20475), with strains having this gene inactivatedbeing of especial interest. For example, in one embodiment, a capsuledeficient strain is generated by disrupting production of a functionalsynX gene product (see, e.g., Swartley et al. (1994) J Bacteriol.176:1530-4).

Capsule-deficient strains can also be generated from naturally-occurringstrains using non-recombinant techniques, e.g., by use of bactericidalanti-capsular antibodies to select for strains with reduced levels ofcapsular polysaccharide.

Where the disclosure involves use of two or more strains (e.g., toproduce antigenic compositions containing a subject fHbp-presentingvesicles from different strains), the strains can be selected so as todiffer in one or more strain characteristics, e.g., to provide forvesicles that differ in the subject fHbp used, PorA, and the like.

Preparation of Vesicles

The antigenic compositions contemplated by the present disclosuregenerally include vesicles prepared from Neisserial cells that express asubject fHbp. As referred to herein “vesicles” is meant to encompassouter membrane vesicles as well as microvesicles (which are alsoreferred to as blebs).

The antigenic composition can contain outer membrane vesicles (OMV)prepared from the outer membrane of a cultured strain of Neisseriameningitidis spp. genetically modified to express a subject fHbp. OMVsmay be obtained from Neisseria meningitidis grown in broth or solidmedium culture, preferably by separating the bacterial cells from theculture medium (e.g. by filtration or by a low-speed centrifugation thatpellets the cells, or the like), lysing the cells (e.g. by addition ofdetergent, osmotic shock, sonication, cavitation, homogenization, or thelike) and separating an outer membrane fraction from cytoplasmicmolecules (e.g. by filtration; or by differential precipitation oraggregation of outer membranes and/or outer membrane vesicles, or byaffinity separation methods using ligands that specifically recognizeouter membrane molecules; or by a high-speed centrifugation that pelletsouter membranes and/or outer membrane vesicles, or the like); outermembrane fractions may be used to produce OMVs.

The antigenic composition can contain microvesicles (MV) (or “blebs”)containing subject fHbps, where the MV or blebs are released duringculture of a Neisseria meningitidis strain genetically modified toexpress a subject fHbp. For example, MVs may be obtained by culturing astrain of Neisseria meningitidis in broth culture medium, separatingwhole cells from the broth culture medium (e.g. by filtration, or by alow-speed centrifugation that pellets only the cells and not the smallerblebs, or the like), and then collecting the MVs that are present in thecell-free culture medium (e.g. by filtration, differential precipitationor aggregation of MVs, or by a high-speed centrifugation that pelletsthe blebs, or the like). Strains for use in production of MVs cangenerally be selected on the basis of the amount of blebs produced inculture (e.g., bacteria can be cultured in a reasonable number toprovide for production of blebs suitable for isolation andadministration in the methods described herein). An exemplary strainthat produces high levels of blebs is described in PCT Publication No.WO 01/34642. In addition to bleb production, strains for use in MVproduction may also be selected on the basis of NspA production, wherestrains that produce higher levels of NspA may be of particular interest(for examples of N. meningitidis strains having different NspAproduction levels, see, e.g., Moe et al. (1999 Infect. Immun 67: 5664).Other strains of interest for use in production of blebs include strainshaving an inactivated GNA33 gene, which encodes a lipoprotein requiredfor cell separation, membrane architecture and virulence (see, e.g.,Adu-Bobie et al. (2004) Infect Immun 72:1914-1919).

The antigenic compositions of the present disclosure can containvesicles from one strain, or from 2, 3, 4, 5 or more strains, whichstrains may be homologous or heterologous, usually heterologous, to oneanother. For example, the strains may be homologous or heterologous withrespect to PorA and/or the fHbp from which the subject fHbp is derived.The vesicles can be prepared from strains that express more than onesubject fHbp (e.g., 1, 2, 3, or more subject fHbp) which may be composedof fHbp amino acid sequences from different variants (v.1, v.2, or v.3)or subvariants (e.g., a subvariant of v.1, v.2, or v.3).

The antigenic compositions can comprise a mixture of OMVs and MVspresenting the same or different subject fHbps, where the subject fHbpsmay optionally present epitopes from different combinations of fHbpvariants and/or subvariants and where the OMVs and/or MVs may be fromthe same or different strains. Vesicles from different strains can beadministered as a mixture, or can be administered serially.

Where desired (e.g., where the strains used to produce vesicles areassociated with endotoxin or particular high levels of endotoxin), thevesicles are optionally treated to reduce endotoxin, e.g., to reducetoxicity following administration. Although less desirable as discussedbelow, reduction of endotoxin can be accomplished by extraction with asuitable detergent (for example, BRIJ-96, sodium deoxycholate, sodiumlauroylsarcosinate, EMPIGEN BB, TRITON X-100, non-ionic detergent TWEEN20 (sorbitan monolaurate polyoxyethylene), non-ionic detergent TWEEN 80,at a concentration of 0.1-10%, e.g., 0.5-2%, and sodium dodecyl sulfate(SDS)). Where detergent extraction is used, it is preferable to use adetergent other than deoxycholate.

The vesicles of the antigenic compositions can be prepared withoutdetergent, e.g., without use of deoxycholate. Although detergenttreatment is useful to remove endotoxin activity, it may deplete thenative fHbp lipoprotein and/or subject fHbp (including lipidated fHbp)by extraction during vesicle production. Thus it may be particularlydesirable to decrease endotoxin activity using technology that does notrequire a detergent. In one approach, strains that are relatively lowproducers of endotoxin (lipopolysaccharide, LPS) are used so as to avoidthe need to remove endotoxin from the final preparation prior to use inhumans. For example, the vesicles can be prepared from Neisseria mutantsin which lipooligosaccharide or other antigens that may be undesirablein a vaccine (e.g. Rmp) is reduced or eliminated.

Vesicles can be prepared from N. meningitidis strains that containgenetic modifications that result in decreased or no detectable toxicactivity of lipid A. For example, such strain can be geneticallymodified in lipid A biosynthesis (Steeghs et al. (1999) Infect Immun67:4988-93; van der Ley et al. (2001) Infect Immun 69:5981-90; Steeghset al. (2004) J Endotoxin Res 10:113-9; Fissha et al, (2005) InfectImmun 73:4070). The immunogenic compositions may be detoxified bymodification of LPS, such as downregulation and/or inactivation of theenzymes encoded by lpxL1 or lpxL2, respectively. Production of apenta-acylated lipid A made in lpxL1 mutants indicates that the enzymeencoded by lpxL1 adds the C12 to the N-linked 3-OH C14 at the 2′position of GlcN II. The major lipid A species found in lpxL2 mutants istetra-acylated, indicating the enzyme encoded by lpxL2 adds the otherC12, i.e., to the N-linked 3-OH C14 at the 2 position of GlcN I.Mutations resulting in a decreased (or no) expression of these genes (ordecreased or no activity of the products of these genes) result inaltered toxic activity of lipid A (van der Ley et al. (2001) InfectImmun 69:5981-90). Tetra-acylated (lpxL2 mutant) and penta acylated(lpxL1 mutant) lipid A are less toxic than the wild-type lipid A.Mutations in the lipid A 4′-kinase encoding gene (lpxK) also decreasethe toxic activity of lipid A. Of particular interest for use inproduction of vesicles (e.g., MV or OMV) are N. meningitidis strainsgenetically modified so as to provide for decreased or no detectablefunctional LpxL1-encoded protein, e.g., where the Neisseria bacterium(e.g., N. meningitidis strain) is genetically modified to provide fordecreased or no activity of a gene product of the lpxL1 gene. Forexample, the Neisseria bacterium can be genetically modified to have anlpxL1 gene knockout, e.g., where the lpxL1 gene is disrupted. See, e.g.,US Patent Publication No. 2009/0035328. The Neisseria bacterium can begenetically modified to provide for decreased or no activity of a geneproduct of the lpxL2 gene. The Neisseria bacterium can be geneticallymodified to provide for decreased or no activity of a gene product ofthe lpxL1 gene and the lpxL2 gene. Such vesicles provide for reducedtoxicity as compared to N. meningitidis strains that are wild-type forLPS production, while retaining immunogenicity of subject fHbp.

LPS toxic activity can also be altered by introducing mutations ingenes/loci involved in polymyxin B resistance (such resistance has beencorrelated with addition of aminoarabinose on the 4′ phosphate of lipidA). These genes/loci could be pmrE that encodes a UDP-glucosedehydrogenase, or a region of antimicrobial peptide-resistance genescommon to many enterobacteriaciae which could be involved inaminoarabinose synthesis and transfer. The gene pmrF that is present inthis region encodes a dolicol-phosphate manosyl transferase (Gunn J. S.,Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. 1998.Mol. Microbiol. 27: 1171-1182).

Mutations in the PhoP-PhoQ regulatory system, which is a phospho-relaytwo component regulatory system (e.g., PhoP constitutive phenotype,PhoPc), or low Mg⁺⁺ environmental or culture conditions (that activatethe PhoP-PhoQ regulatory system) lead to the addition of aminoarabinoseon the 4′-phosphate and 2-hydroxymyristate replacing myristate(hydroxylation of myristate). This modified lipid A displays reducedability to stimulate E-selectin expression by human endothelial cellsand TNF secretion from human monocytes.

Polymyxin B resistant strains are also suitable for use, as such strainshave been shown to have reduced LPS toxicity (see, e.g., van der Ley etal. (1994) In: Proceedings of the ninth international pathogenicNeisseria conference. The Guildhall, Winchester, England).Alternatively, synthetic peptides that mimic the binding activity ofpolymyxin B may be added to the antigenic compositions to reduce LPStoxic activity (see, e.g., Rustici et al. (1993) Science 259:361-365;Porro et al. (1998) Prog Clin Biol Res. 397:315-25).

Endotoxin can also be reduced through selection of culture conditions.For example, culturing the strain in a growth medium containing 0.1mg-100 mg of aminoarabinose per liter medium provides for reduced lipidtoxicity (see, e.g., WO 02/097646).

Compositions and Formulations

“Compositions”, “antigen composition”, “antigenic composition” or“immunogenic composition” is used herein as a matter of convenience torefer generically to compositions comprising a subject fHbp as disclosedherein, which subject fHbp may be optionally conjugated to furtherenhance immunogenicity. Compositions useful for eliciting antibodies,e.g., antibodies against Neisseria meningitidis, e.g., bactericidalantibodies to Neisseria meningitidis, in a human are specificallycontemplated by the present disclosure. Antigenic compositions cancontain 1, 2, or more different subject fHbps. Where there is more thanone type of fHbp, each subject fHbps may present epitopes from differentcombinations of fHbp variants and/or subvariants.

Antigenic compositions contain an immunologically effective amount of asubject fHbp, and may further include other compatible components, asneeded. Compositions of the present disclosure can contain fHbp that arelow fH binders. The composition contain one or more fHbp, in which atleast one fHbp is a low fH binder. Where there is more than one fHbp ina composition, each fHbp may be different (e.g. in amino acid sequencesand/or conjugation).

In some cases, an antigenic composition of the present disclosurecomprises only one fHbp variant of the present disclosure. In somecases, an antigenic composition of the present disclosure comprises twoor more different fHbp variants of the present disclosure. Asnon-limiting examples, in some cases, an antigenic composition of thepresent disclosure comprises:

1) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at Q38 (e.g., Q38R); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at E92 (e.g., E92K);

2) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at Q38 (e.g., Q38R); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at R130 (e.g., R130G);

3) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at Q38 (e.g., Q38R); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at S223 (e.g., S223R);

4) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at Q38 (e.g., Q38R); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at H248 (e.g., H248L);

5) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at N115 (e.g., N115I); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atQ38 (e.g., Q38R);

6) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at D121 (e.g., D121G); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atE92 (e.g., E92K);

7) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at 5128 (e.g., S128T); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atH248 (e.g., H248L);

8) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at V131 (e.g., V131D); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atQ38 (e.g., Q38R);

9) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at K219 (e.g., K219N); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atQ38 (e.g., Q38R);

10) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S); and a variant of fHbp ID1, where the fHbp ID 1 variant comprises an amino acid substitution atQ38 (e.g., Q38R);

11) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at N115 (e.g., N115I); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atE92 (e.g., E92K);

12) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at D121 (e.g., D121G); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atS223 (e.g., S223R);

13) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at 5128 (e.g., S128T); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atH248 (e.g., H248L);

14) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at V131 (e.g., V131D); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atE92 (e.g., E92K);

15) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at K219 (e.g., K219N); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atE92 (e.g., E92K);

16) a variant of fHbp ID 22, where the fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S); and a variant of fHbp ID55, where the fHbp ID 55 variant comprises an amino acid substitution atE92 (e.g., E92K);

17) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at E92 (e.g., E92K); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at H248 (e.g., H248L);

18) a first variant of fHbp ID 1, where the first fHbp ID 1 variantcomprises an amino acid substitution at E92 (e.g., E92K); and a secondvariant of fHbp ID 1, where the second fHbp ID 1 variant comprises anamino acid substitution at S223 (e.g., S223R);

19) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at N115 (e.g., N115I); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at D211 (e.g., D211A);

20) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at N115 (e.g., N115I); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at K219 (e.g., K219N);

21) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at N115 (e.g., N115I); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S);

22) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at D121 (e.g., D121G); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S);

23) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at 5128 (e.g., S128T); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S);

24) a first variant of fHbp ID 22, where the first fHbp ID 22 variantcomprises an amino acid substitution at V131 (e.g., V131D); and a secondvariant of fHbp ID 22, where the second fHbp ID 22 variant comprises anamino acid substitution at G220 (e.g., G220S);

25) a first variant of fHbp ID 55, where the first fHbp ID 55 variantcomprises an amino acid substitution at E92 (e.g., E92K); and a secondvariant of fHbp ID 55, where the second fHbp ID 55 variant comprises anamino acid substitution at S223 (e.g., S223R);

26) a first variant of fHbp ID 55, where the first fHbp ID 55 variantcomprises an amino acid substitution at E92 (e.g., E92K); and a secondvariant of fHbp ID 55, where the second fHbp ID 55 variant comprises anamino acid substitution at H248 (e.g., H248L);

27) a variant of fHbp ID22 comprising amino acid substitutions: L130Rand G133D and a variant of fHbp ID 1, where the fHbp ID 1 variantcomprises an amino acid substitution at S223 (e.g., S223R);

28) a variant of fHbp ID22 comprising amino acid substitutions: L130Rand G133D and a variant of fHbp ID 1, where the fHbp ID 1 variantcomprises an amino acid substitution at H248 (e.g., H248L);

29) a variant of fHbp ID22 comprising amino acid substitutions: L130R,G133D, and K219N and a variant of fHbp ID 1, where the fHbp ID 1 variantcomprises an amino acid substitution at S223 (e.g., S223R) or H248(e.g., H248L); or

30) a variant of fHbp ID22 comprising amino acid substitutions: L130R,G133D, and G220S and a variant of fHbp ID 1, where the fHbp ID 1 variantcomprises an amino acid substitution at S223 (e.g., S223R) or H248(e.g., H248L).

Immunogenic compositions contemplated by the present disclosure include,but are not limited to, compositions comprising:

1) at least one variant fHbp of the present disclosure; and

2) NspA;

where the fHbp and/or NspA can be provided as recombinant proteinsand/or in a vesicle-based composition (e.g., OMV or MV). It should benoted that where the composition includes both NspA and a fHbp, thebactericidal activity of antibodies elicited by administration of thecomposition can result from cooperation of antibodies to one or bothantigens. Examples of immunogenic compositions provided by the presentdisclosure include:

a) an immunogenic composition that comprises a fHbp variant as describedabove (e.g., where the variant fHbp elicits a bactericidal antibodyresponse to at least one Neisseria meningitidis strain);

b) an immunogenic composition that comprises a fHbp variant as describedabove (e.g., where the variant fHbp elicits a bactericidal antibodyresponse to at least one Neisseria meningitidis strain); and arecombinant NspA protein;

c) an immunogenic composition that comprises a native OMV obtained froma genetically modified Neisseria host cell that is genetically modifiedwith a nucleic acid encoding a variant fHbp of the present disclosure,such that the encoded variant fHbp is produced by the geneticallymodified host cell, where the OMV comprises the encoded variant fHbp;and

d) an immunogenic composition that comprises a native OMV obtained froma genetically modified Neisseria host cell that is genetically modifiedwith a nucleic acid encoding a variant fHbp of the present disclosure,such that the encoded non-naturally occurring fHbp is produced by thegenetically modified host cell, where the OMV comprises the encodedvariant fHbp; and where the Neisseria host cell also produces higherlevels of NspA, such that the OMV also comprises NspA. For example, theNeisseria host cell can be one that is genetically modified forincreased expression of NspA.

By “immunologically effective amount” is meant that the administrationof that amount to an individual, either in a single dose, as part of aseries of the same or different antigenic compositions, is effective toelicit an antibody response effective for treatment or prevention of asymptom of, or disease caused by, for example, infection by Neisseria,particularly N. meningitidis, more particularly Group B N. meningitidis.This amount varies depending upon the health and physical condition ofthe individual to be treated, age, the capacity of the individual'simmune system to produce antibodies, the degree of protection desired,the formulation of the vaccine, the treating clinician's assessment ofthe medical situation, and other relevant factors. It is expected thatthe amount will fall in a relatively broad range that can be determinedthrough routine trials.

Amino acid sequences of NspA polypeptides are known in the art. See,e.g., WO 96/29412; and Martin et al. (1997) J. Exp. Med. 185:1173;GenBank Accession No. U52066; and GenBank Accession No. AAD53286. An“NspA polypeptide” can comprise an amino acid sequence having at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 75 amino acids to about100 amino acids, from about 100 amino acids to about 150 amino acids orfrom about 150 amino acids to about 174 amino acids, of the amino acidsequence depicted in FIG. 40 and set forth in SEQ ID NO:25. An “NspApolypeptide” can comprise an amino acid sequence having at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 75 amino acids to about100 amino acids, or from about 100 amino acids to about 155 amino acids,of amino acids 20 to 174 of the amino acid sequence depicted in FIG. 40and set forth in SEQ ID NO:25. In some cases, the NspA polypeptide lacksa signal sequence; in other cases (e.g., for expression in a host cell),the NspA polypeptide includes a signal sequence.

Dosage regimen may be a single dose schedule or a multiple dose schedule(e.g., including booster doses) with a unit dosage form of the antigeniccomposition administered at different times. The term “unit dosageform,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of the antigenic compositions of the presentdisclosure in an amount sufficient to produce the desired effect, whichcompositions are provided in association with a pharmaceuticallyacceptable excipient (e.g., pharmaceutically acceptable diluent, carrieror vehicle). The antigenic composition may be administered inconjunction with other immunoregulatory agents.

Antigenic compositions can be provided in a pharmaceutically acceptableexcipient, which can be a solution such as a sterile aqueous solution,often a saline solution, or they can be provided in powder form. Suchexcipients can be substantially inert, if desired.

In some embodiments, a subject immunogenic composition comprises asubject fHbp present in a vesicle. In some embodiments, a subjectimmunogenic composition comprises a subject fHbp present in an MV. Insome embodiments, a subject immunogenic composition comprises a subjectfHbp present in an OMV. In some embodiments, a subject immunogeniccomposition comprises a mixture of MV and OMV comprising a subject fHbp.Vesicles, such as MV and OMV, are described above.

The antigenic compositions can further contain an adjuvant. Examples ofknown suitable adjuvants that can be used in humans include, but are notnecessarily limited to, an aluminum adjuvant (e.g., aluminum phosphate,or aluminum hydroxide), MF59 (4.3% w/v squalene, 0.5% w/v TWEEN 80™,0.5% w/v SPAN 85), a CpG-containing nucleic acid (where the cytosine isunmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CTmutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, and the like. For experimental animals, one can useFreund's adjuvant (incomplete Freund's adjuvant; complete Freund'sadjuvant), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicantigen or antigenic epitope thereof.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunitand adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% TWEEN 80, and 0.5% SPAN 85 (optionallycontaining MTP-PE) formulated into submicron particles using amicrofluidizer, (b) SAF, containing 10% Squalane, 0.4% TWEEN 80, 5%PLURONIC-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween TWEEN 80, and one ormore bacterial cell wall components such as monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g., MPL+CWS(Detox TM); (2) saponin adjuvants, such as QS21 or Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes), which ISCOMS maybe devoid of additional detergent e.g. WO 00/07621; (3) CompleteFreund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA); (4)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (W099/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (see, e.g., WO 98/52581),e.g., an oligonucleotide containing at least one CG dinucleotide, wherethe cytosine is unmethylated; (8) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g. WO 99/52549); (9) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO 01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g.a CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and aparticle of metal salt e.g. WO 00/23105; (12) a saponin and anoil-in-water emulsion e.g. WO 99/11241; (13) a saponin (e.g.QS21)+3dMPL+IM2 (optionally+a sterol) e.g. WO 98/57659; (14) othersubstances that act as immunostimulating agents to enhance the efficacyof the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl—sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Adjuvants suitable for administration to a human are ofparticular interest. In some cases, the adjuvant is an aluminum saltadjuvant (e.g., aluminum phosphate or aluminum hydroxide).

The antigen compositions may contain other components, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of the subject fHbp in a formulation can vary widely(e.g., from less than about 0.1%, e.g., at or at least about 2% to asmuch as 20% to 50% or more by weight) and will usually be selectedprimarily based on fluid volumes, viscosities, and patient-based factorsin accordance with the particular mode of administration selected andthe patient's needs.

The fHbp-containing formulations can be provided in the form of asolution, suspension, tablet, pill, capsule, powder, gel, cream, lotion,ointment, aerosol or the like. It is recognized that oral administrationcan require protection of the compositions from digestion. This istypically accomplished either by association of the composition with anagent that renders it resistant to acidic and enzymatic hydrolysis or bypackaging the composition in an appropriately resistant carrier. Meansof protecting from digestion are well known in the art.

The fHbp-containing formulations can also be provided so as to enhanceserum half-life of fHbp following administration. For example, whereisolated fHbps are formulated for injection, the fHbp may be provided ina liposome formulation, prepared as a colloid, or other conventionaltechniques for extending serum half-life. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028. The preparations may also be provided incontrolled release or slow-release forms.

Methods of Inducing an Immune Response

The present disclosure provides a method of inducing an immune responseto at least one Neisserial strain in a mammalian host. The methodsgenerally involve administering to an individual in need thereof aneffective amount of a subject immunogenic composition.

The fHbp-containing antigenic compositions are generally administered toa human subject that is at risk of acquiring a Neisserial disease so asto prevent or at least partially arrest the development of disease andits complications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for therapeutic usewill depend on, e.g., the antigenic composition, the manner ofadministration, the weight and general state of health of the patient,and the judgment of the prescribing physician. Single or multiple dosesof the antigenic compositions may be administered depending on thedosage and frequency required and tolerated by the patient, and route ofadministration.

The fHbp-containing antigenic compositions are generally administered inan amount effective to elicit an immune response, particularly a humoralimmune response, e.g., a bactericidal antibody response, in the host. Asnoted above, amounts for immunization will vary, and can generally rangefrom about 1 μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg/70kg. Substantially higher dosages (e.g. 10 mg to 100 mg or more) may besuitable in oral, nasal, or topical administration routes. The initialadministration can be followed by booster immunization of the same ofdifferent fHbp-containing antigenic composition. Vaccination in somecases involves at least one booster, and in some cases two boosters.

In general immunization can be accomplished by administration by anysuitable route, including administration of the composition orally,nasally, nasopharyngeally, parenterally, enterically, gastrically,topically, transdermally, subcutaneously, intramuscularly, in tablet,solid, powdered, liquid, aerosol form, locally or systemically, with orwithout added excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

An anti-fHbp immune response can be assessed by known methods (e.g. byobtaining serum from the individual before and after the initialimmunization, and demonstrating a change in the individual's immunestatus, for example an immunoprecipitation assay, an ELISA, or abactericidal assay, a Western blot assay, or flow cytometric assay, orthe like).

Whether a variant fHbp of the present disclosure elicits a bactericidalresponse to one or more strains of N. meningitidis in a mammalian hostcan be determined using any well-known assay. For example, a human fHtransgenic mouse can be used, where the mouse expresses human fH (e.g.,human fH is present in serum of the mouse at a concentration of about100 μg/ml or greater than 100 μg/ml). A variant fHbp of the presentdisclosure is administered to the human fH transgenic mouse. After aperiod of time, serum from the mouse is tested for bactericidal activityagainst one or more strains of N. meningitidis. Suitable controlsinclude, e.g., fHbp ID 1. An example of a suitable assay is described inVu et al. (2012) Sci. Reports 2:341.

The antigenic compositions can be administered to a mammalian host(e.g., a human subject) that is immunologically naive with respect toNeisseria meningitidis. In a particular embodiment, the subject is ahuman child about five years or younger, and preferably about two yearsold or younger, and the antigenic compositions are administered at anyone or more of the following times: two weeks, one month, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 months, or one year or 15, 18, or 21 months afterbirth, or at 2, 3, 4, or 5 years of age.

It may be generally desirable to initiate immunization prior to thefirst sign of disease symptoms, or at the first sign of possible oractual exposure to infection or disease (e.g., due to exposure orinfection by Neisseria).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Identification and Characterization of fHbp ID 1 Mutants

Materials and Methods

Library Screening

A random mutant fHbp library was generated by error-prone polymerasechain reaction (PCR), which was followed by cloning of the PCR productsinto a pET28 expression plasmid that included a signal sequence to allowsurface display on E. coli. Fluorescence-activated cell sorting was usedto isolate mutant clones with low binding of human fH and high bindingof a control anti-fHbp monoclonal antibody, which ensured sufficientexpression and proper folding of the fHbp mutants. The collected cellswere plated on agar plates (LB agar containing 50 μg/ml kanamycinsulfate), which were incubated overnight at 37° C. Single E. colicolonies were used as templates for PCR amplification and the DNAamplicons were purified (PCR Purification Kit; Qiagen) and subjected toDNA sequencing of the fHbp gene using primers that annealed to the T7promotor and T7 terminator. The approach of screening a random mutantfHbp library has the potential to identify: 1) positions that affect fHbinding that are not predictable from the crystal structure alone; and2) substitutions other than alanine that affect binding of fH in caseswhere an alanine substitution would not result in a sufficient decreasein fH binding.

Selection of Mutants for Further Study

The positions of amino acid substitutions that were identified from theFACS experiment were examined in the crystal structure of fHbp in acomplex with a fragment of human fH. Mutants that were in proximity (<5Å) to the fH binding interface were chosen for site-specificmutagenesis. Recapitulation of the library mutants by site-specificmutagenesis was necessary to create soluble, recombinant fHbp proteinsfor further characterization. This approach also removed non-desiredsecondary mutations, which were present in many of the sorted clones andwhich were distant from the fH binding site. The site-specific mutantswere constructed with the Phusion Site-Directed Mutagenesis Kit (ThermoScientific, Inc.).

Expression and Purification of Soluble, Mutant fHbps

Soluble, recombinant fHbps were expressed in E. coli and lysates wereprepared as previously described. fHbps were purified by nickel-affinitychromatography using HiTrap Chelating HP columns (5 ml; GE LifeSciences, Inc.) and an Akta Purifier chromatography system (GE LifeSciences). Buffers for binding and elution using an imidazole gradientwere prepared according to the column manufacturer's protocols.Fractions containing purified fHbp were combined, dialyzed against PBScontaining 3% sucrose and stored at −80° C. prior to use.

For mouse immunogenicity studies, a second purification step wasperformed using ion exchange chromatography with HiTrap SP HP columns (5ml; GE Life Sciences). The binding and elution buffers were 25 mM MES,pH 5.5, containing 150 mM and 750 mM NaCl, respectively. Bound fHbp waseluted from the SP column with a linear gradient formed by the bindingand elution buffers. Fractions containing purified fHbp were combined,dialyzed against PBS containing 3% sucrose and stored at −80° C. priorto use.

Purification of Human Factor H (fH)

Human fH was purified on a fHbp affinity column. The column was preparedby coupling 5 mg of fHbp ID 1 to an NHS-activated HP column (5 ml; GELife Sciences) using the manufacturer's protocols. Human serum from ahealthy individual donor was diluted 1:1 in phosphate-buffered saline(PBS). The serum was applied to the column and the column was washedwith 10 volumes of PBS (i.e. 50 ml). The bound fH was eluted with 5column volumes of 0.1 M glycine-HC1, pH 2.7. The elution fractions werecollected in tubes containing 50 μl of 1 M TRIS-HCl, pH 9.0. Fractionscontaining fH were identified by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) (4-12% NuPAGE; Invitrogen).Electrophoresis was performed at 200 V for 45 min using 1× MES RunningBuffer (Invitrogen). The proteins were visualized by staining withCOOMASSIE G-250 (SimplyBlue SafeStain; Invitrogen). Fractions containingfH were pooled and dialyzed against PBS and aliquots of fH were storedat −30° C. prior to use.

Characterization of fHbp Mutants

SDS-PAGE. The size and purity of the purified fHbp mutant proteins wasassessed by SDS-PAGE using 4-12% polyacrylamide gradient gels (NuPAGE;Invitrogen, Inc.). Two μg of each protein was loaded on the gel.SDS-PAGE was performed as described above for fH.

Binding of fH to fHbp by enzyme-linked immunosorbent assay (ELISA). Thewells of a 96-well microtiter plate (Immulon 2HB; Thermo Scientific)were coated with 2 μg/ml of purified, recombinant wild-type fHbp(positive control) or mutant fHbp (experimental). Non-specific bindingto the wells was blocked with PBS containing 1% BSA (Lifeblood Medical,Inc.) or 5% non-fat dry milk (Carnation; Nestle, Inc.). Five-fold serialdilutions of purified human fH ranging from 25 to 0.0016 μg/ml inDilution Buffer (PBS containing 0.1% Tween-20, 0.01% sodium azide and 1%BSA), were added to the wells and the plate was incubated at roomtemperature for 2 h. After washing three times with PBS containing 0.1%Tween-20 (Sigma) and 0.01% sodium azide (Sigma), bound fH was detectedwith sheep anti-human fH (1:7,000; Abcam, Inc.) in Dilution Buffer. Theplate was incubated at room temperature for 1 h. After washing the wellsagain, bound primary antibody was detected with donkey anti-sheep IgGconjugated to alkaline phosphatase (1:5,000; Sigma-Aldrich, Inc.) inDilution Buffer. The plate was incubated at room temperature for 1 h andthe wells were washed again. The ELISA was developed with phosphatasesubstrate (1 mg/ml para-nitrophenyl phosphate; Sigma) in SubstrateBuffer (50 mM sodium carbonate, 1 mM MgCl₂, pH 9.8). After incubation atroom temperature for 30 min, the absorbance at 405 nm was measured in aUV-VIS plate reader (Spectromax 190; Molecular Devices, Inc.).

Binding of anti-fHbp monoclonal antibodies to fHbp by ELISA. The wellsof a microtiter plate were coated with fHbp, blocked and washed asdescribed above for the fH ELISA. Five-fold serial dilutions of murineanti-fHbp monoclonal antibodies (mAbs) from 25 to 0.0016 μg/ml inDilution Buffer were added and the plate was incubated at roomtemperature for 1 h. After washing the wells, primary antibody wasdetected with goat anti-mouse IgG conjugated to alkaline phosphatase(1;5,000; Sigma-Aldrich). The ELISA was developed and read as describedabove.

Binding of fH to fHbp by surface plasmon resonance (SPR). SPRexperiments were performed on a Biacore X100 Plus instrument (GE LifeSciences). Three thousand response units of purified human fH werecoupled to a CM5 chip (GE Life Sciences) using the Amine Coupling Kit(GE Life Sciences). fH was immobilized in flow cell 2 and a blankimmobilization (no fH) was performed in flow cell 1 as a reference.Three startup cycles consisting of HEPES-buffered saline containing 3 mMEDTA and 0.05% Surfactant P-20 (GE Life Sciences) and regeneration with100 mM Glycine, 3 M NaCl, pH 2.0, were performed to condition the chipsurface. Dilutions of purified, recombinant fHbp ranging from 100 to 1nM (wild-type) or 316 to 3.16 nM were injected for 150 seconds.Dissociation was monitored for 300 sec. and the data were analyzed withBiacore X100 Evaluation software.

Mouse immunogenicity. Groups of wild-type CD-1 mice (N=14 to 21) wereimmunized with fHbp vaccines adsorbed with aluminum hydroxide. Each doseof vaccine contained 10 μg of fHbp and 600 μg of ALHYDROGEL (BrenntagBiosector) in 10 mM Histidine, 150 mM NaCl, pH 6.5. Two doses were giventhree-weeks apart and blood was collected by cardiac puncture threeweeks after the second dose. Blood was processed to obtain serum, whichwas kept at −80° C. for long-term storage (>2 weeks) or 4° C. forshort-term storage (<2 weeks).

Human fH transgenic BALB/c mice first were screened to identify animalswith serum human fH concentrations >240 μg/ml using a fHbp ELISA and astandard curve of purified human fH. The ELISA was performed usingpurified fHbp ID 1 immobilized on the plate, and the primary andsecondary antibodies to detect fH were the same as described above (see“Binding of fH to fHbp by ELISA”).

Groups of transgenic mice (N=11 to 21) were immunized with fHbp vaccinesadsorbed with aluminum hydroxide (same amount of antigen and adjuvant asfor wild-type CD-1 mice described above). Three doses were administeredat three-week intervals and blood was collected three weeks after thethird dose of vaccine. Serum was processed and stored as describedabove.

Serum bactericidal antibody (SBA) responses. Human complement-mediatedSBA responses were measured against meningococcal strains with anidentical or closely matched fHbp sequence compared with the respectivevaccine antigen. The bacteria were grown in regular Frantz medium(Frasch et al. “Outer membrane protein vesicle vaccines formeningococcal disease.” In Methods in Molecular Medicine, v. 66.Meningococcal Vaccines: Methods and Protocols. Edited by Pollard, A. J.and Maiden, M. C. Humana Press Inc. Totowa, N.J.) containing 4 mMlactate and 0.02 mM CMP-NANA to mid-exponential phase (OD620 nm=0.6).The bacteria were diluted 1:25,000 in Dulbecco's PBS containing 1% BSA(Equitech Bio.). Human complement was from a donor with no intrinsicbactericidal antibodies and was depleted of IgG antibodies using aHiTrap Protein G column (5 ml; GE Life Sciences). Each reactioncontained 25% human complement, ˜400 cfu of bacteria and dilutions oftest antisera or control antibodies. The SBA titer was calculated as theserum dilution that resulted in a 50% decrease in cfu relative tonegative control wells after 60 min incubation at 37° C.Proteinpurification: Recombinant fHbps were expressed in E. coli with aC-terminal hexa-histidine tag and purified by metal chelatechromatography (HiTrap Chelating HP; GE Life Sciences) followed by ionexchange chromatography (HiTrap SP; GE Life Sciences). The proteins (2μg each) were separated on a 4-12% NuPAGE gel (Invitrogen) using MESrunning buffer (Invitrogen), and visualized with COOMASSIE blue staining(Simply Blue Safe Stain; Invitrogen).

Results

A random mutant library-based approach was developed to identify fHbpmutants with decreased binding of human fH. This approach was able toidentify mutations leading to decreased binding of fH that might not bepredictable based on structural information alone, and was able togenerate multiple amino acid substitutions at any given position. Thisapproach contrasts with the common approach of substitution of alanineat selected positions, which sometimes results in small decreases inbinding of fH.

Using the random mutant library approach, we identified five promisingnew fHbp ID 1 mutants. The purified, recombinant mutant fHbp ID 1antigens Q38R, E92K, R130G, S223R and H248L are shown in FIG. 1.

FIG. 1. Purity of fHbp ID 1 mutants. Recombinant fHbps were expressed inE. coli with a C-terminal hexa-histidine tag and purified by metalchelate chromatography (HiTrap Chelating HP; GE Life Sciences) followedby ion exchange chromatography (HiTrap SP; GE Life Sciences). Theproteins (2 μg each) were separated on a 4-12% NuPAGE gel (Invitrogen)using MES running buffer (Invitrogen), and visualized with COOMASSIEblue staining (Simply Blue Safe Stain; Invitrogen). Lane 1, Kaleidoscopemolecular weight marker (Bio-Rad Laboratories); 2, fHbp ID 1 wild-type;3, Q38R; 4, E92K; 5, R130G; 6, S223R; 7, H248L.

These mutants exhibit decreased binding of fH ranging from ˜10-fold(R130G) to ˜20-fold (Q38R) to ˜100-fold (E92K, S223R and H248L) (FIG.2).

FIGS. 2A and 2B. fH binding of fHbp ID 1 mutants by ELISA. The wells ofa microtiter plate were coated with purified recombinant ID 1 wild-type(WT) or one of six different mutant proteins. Different concentrationsof purified human fH were added to the wells. Bound fH was detected withsheep anti-human fH (Abcam) and donkey anti-sheep IgG conjugated toalkaline phosphatase (Sigma). A, Positive control fHbp ID 1 wild-type(WT) protein with high binding of human fH. Negative control fHbp ID 1R41S mutant with low binding of fH. B, New fHbp ID 1 mutants withdecreased binding of fH. The R130G mutant showed moderate binding of fH,Q38R showed low binding and E92K, S223R and H248L showed significantlylower binding than that of R41S. The mean and standard deviation forreplicate measurements are shown.

A similar pattern of decreased binding of fH to the mutant proteins wasobtained from surface plasmon experiments, in which the R130G and Q38Rmutants show some binding, whereas the other three mutants show nodetectable binding (FIGS. 3A and 3B).

FIGS. 3A-3E. fH binding of fHbp ID 1 mutants by surface plasmonresonance. 3000 response units of purified human fH were coupled to aCM5 chip (GE Life Sciences) and 316 nM of purified, recombinant fHbp wasinjected for 150 seconds. For reference, the same data for the ID 1wild-type (WT) protein are shown in each panel. The same pattern ofbinding was observed as in the ELISA (FIG. 2, above); moderate bindingto fH for R130G mutant, low binding for Q38R and very low binding forE92K, S223R and H248L. All experiments employed HBS-EP running bufferand a Biacore X100 Plus surface plasmon resonance instrument. Data wereanalyzed with Biacore X100 Evaluation software.

All five of the mutant fHbp ID 1 proteins retained conformationalepitopes recognized by anti-fHbp monoclonal antibodies.Concentration-dependent binding of five anti-fHbp monoclonal antibodiesto wild-type or mutant fHbps is shown in FIG. 4.

FIGS. 4A-4E. Binding of murine anti-fHbp monoclonal antibodies to fHbpmutant proteins as measured by ELISA. Similar concentration-dependentbinding of anti-fHbp monoclonal antibodies indicated that the wild-typeand mutant fHbps were present in similar amounts in the wells of themicrotiter plate and that the mutant fHbps retained conformationalepitopes recognized by five distinct monoclonal antibodies. Thesecondary antibody was goat anti-mouse IgG conjugated to alkalinephosphatase (Sigma). The mean and standard deviation for duplicatemeasurements are shown.

The mutant proteins also retain thermal stability similar to thewild-type fHbp ID 1, except the E92K mutant, which has somewhatdecreased stability. Finally, the mutants elicited similar bactericidalantibody responses in wild-type CD-1 mice when tested against serogroupB strain H44/76 (FIGS. 5A-5B).

FIGS. 5A and 5B. Bactericidal antibody responses to fHbp ID 1 mutants inmice. FIG. 5A, Groups of 12 to 14 wild-type mice were immunizedintraperitoneally with two doses of purified recombinant fHbp (10 μg perdose) given at three-week intervals. Serum was obtained three weeksafter the second dose. Serum bactericidal activity was measured usingIgG-depleted human serum as the complement source and serogroup B strainH44/76 as the test strain. H44/76 expresses fHbp ID 1, which matches thecontrol fHbp ID 1 WT vaccine. Each symbol represents the titer of anindividual mouse, and the horizontal bars represent the geometric meantiters. The differences between the WT group and each of the mutantgroups were not statistically significantly different (p>0.4 by t-test).FIG. 5B, Groups of 14 to 15 human fH transgenic mice were immunizedintraperitoneally with three doses of purified recombinant fHbp (10 μgper dose) given at three-week intervals. Serum was obtained three weeksafter the third dose. Serum bactericidal activity was measured asdescribed above for wild-type mice.

FIGS. 6A and 6B. Binding of human fH to fHbp ID 1 single and doublemutants by ELISA. The experiments were performed as described above forFIGS. 2A-2B. FIG. 7. Bactericidal antibody responses to fHbp ID 1 singleand double mutants in mice. Groups of 20 wild-type mice were immunizedand the serum bactericidal antibody responses were determined asdescribed above for FIG. 5A.

Example 2: Characterization of fHbp ID 55 Mutants

Materials and Methods

The experiments were performed as described in Example 1.

Results

Three promising mutants that were identified in fHbp ID 1 also wereconstructed in fHbp ID 55; these included E92K, S223R and H248L. Allthree of the fHbp ID 55 mutants had significantly decreased binding offH (FIG. 8A). The mutants had preserved conformational integrity asjudged by binding of murine anti-fHbp monoclonal antibody JAR 41 (FIG.8B).

FIGS. 8A and 8B. fH binding of fHbp ID 55 mutants. A. Binding of fH toimmobilized fHbp ID 55 mutants by ELISA. The experiment was performed asdescribed in the legend to FIG. 2. The mean and range for two to fourreplicates are shown. B. Concentration dependent binding of anti-fHbpmonoclonal antibody (mAb) JAR 41 indicated that the recombinant fHbpswere present in similar amounts in the wells of the microtiter plate andhad preserved conformation in the region of the epitope recognized byJAR 41 (N-terminal domain; Vu et al. (2012) Sci. Reports, supra). Thesecondary antibody was goat anti-mouse IgG conjugated to alkalinephosphatase (Sigma).

FIG. 9. Bactericidal antibody responses to fHbp ID 55 mutants inwild-type mice. Groups of 12 mice were immunized and the serumbactericidal antibody responses were determined as described above forFIG. 5A. Bactericidal activity was measured against a mutant of strainH44/76 that expresses fHbp ID 55.

FIG. 10A, Bactericidal antibody responses of human fH transgenic mice tofHbp ID 55 S223R mutant. Groups of 11 to 12 transgenic mice wereimmunized with three doses of purified recombinant fHbp (12 μg per dose)or one-tenth of a human dose of the licensed Trumenba (Pfizer) vaccinecontaining a total of 12 μg fHbp. The transgenic mice were immunized andthe serum bactericidal antibody responses were determined as describedabove for FIG. 5B. FIG. 10B, Bactericidal antibody responses to Trumenbain wild-type and human fH transgenic mice in relation to serum human fHconcentrations. Serum human fH concentrations were measured by ELISA asdescribed previously (Beernink et al. (2011) Journal of Immunology186(6):3606-14).

Example 3: Identification and Characterization of fHbp ID 22 Mutants

Materials and Methods

The experiments were performed as described in Example 1.

Results

An independent search for random fHbp mutants with decreased binding offH was performed using fHbp ID 22, which is in variant group 2(sub-family A). This screen resulted in six promising new mutants (FIG.17). fH binding to the ID 22 wild-type and previously described D211Amutant by ELISA is shown in FIG. 11A. fH binding to the six new mutantID 22 proteins is shown in FIG. 11B.

FIGS. 11A-11D. fH binding of fHbp ID 22 library mutants. FIG. 11A,Positive control fHbp ID 22 wild-type (WT) protein with high binding ofhuman fH. Negative control fHbp ID 22 D211A mutant with low binding offH. FIG. 11B, New fHbp ID 22 mutants with decreased binding of fH. Allof the mutants showed low binding and V131D showed very low bindingsimilar to D211A. The experiment was performed as described in thelegend to FIG. 2. The mean and range of two to four replicates areshown. FIG. 11C, fH binding of a subset of fHbp ID 22 mutants at fHconcentrations up to 100 μg/ml. FIG. 11D, Binding of anti-fHbpmonoclonal antibody JAR 4 to a subset of the mutants (same symbols usedas in FIG. 11C). New mutant K219N retains JAR 4 binding, whereas G220Smutant has decreased binding of JAR 4. All of the mutants had normalbinding of another anti-fHbp monoclonal antibody JAR 31 (data notshown). The mean and standard deviation of duplicate measurements areshown.

Bactericidal antibody responses of wild-type CD-1 mice to the newmutants V131D and K219N, along with the control wild-type ID 22 proteinand the previously characterized mutant D211A are shown in FIG. 12.

FIGS. 12A-12B. Bactericidal antibody responses to fHbp ID 22 librarymutants in wild-type mice. Library mutants with low binding of human fHwere selected for immunization. Groups of 10 to 21 mice were immunizedwith two doses of purified recombinant fHbp (10 μg per dose) given atthree-week intervals. Serum was obtained three weeks after the seconddose. Serum bactericidal activity was measured using IgG depleted humanserum as the complement source and serogroup B strain CH597 as the teststrain. This strain expresses fHbp ID 23, which closely matches thecontrol fHbp ID 22 WT vaccine. FIG. 12A, Experiment testing new mutantsV131D and K219N. The D211A mutant was used as a control mutant fHbpvaccine that did not decrease immunogenicity. Each symbol represents thetiter of an individual mouse, and the horizontal bars represent thegeometric mean titers. FIG. 12B, Second experiment testing new mutantsD121G, S128T, F129S, and G220S. No significant loss of immunogenicitywas observed for the new mutant fHbps, except for a modest loss forV131D.

FIG. 13. Bactericidal antibody responses to fHbp ID 22 library mutantK219N in human fH transgenic mice. The control mutant D211A gave higherresponses and the K219N mutant gave responses similar to the fHbp ID 22wild-type (WT) antigen.

FIG. 14. Thermal stability of fHbp ID 22 measured by differentialscanning microcalorimetry. The fHbp ID 22 wild-type (WT, solid line)undergoes unfolding transitions at 38 (N-terminal) and 81° C.(C-terminal domain) An fHbp ID 22 L130R/G133D double mutant exhibits 19°C. higher thermal stability for the N-terminal domain compared with thatof the ID 22 WT.

FIGS. 15A-15B. fHbp ID 22 triple mutants combining stabilizingsubstitutions L130R and G133D (double mutant, DM) with library derivedmutants to decrease fH binding. A, fH binding to fHbp ID 22 triplemutants. B, Control murine anti-fHbp monoclonal antibody (mAb) JAR 4binding (same symbols as in panel A. All of the stabilized mutants bindJAR 4 better than the fHbp ID 22 WT.

FIG. 16. Bactericidal antibody responses to fHbp ID 22 triple mutants inhuman fH transgenic mice. The two mutants tested combine stabilitydouble mutant (DM) with K219N and G220S, respectively. The triplemutants elicited eight- and 18-fold higher responses than the control ID22 WT antigen.

A summary of exemplary fHbp ID 1 and ID 22 mutants described above ispresented in the table in FIG. 17.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A variant factor H binding protein (fHbp),wherein the variant fHbp comprises: the amino acid sequence of fHbp ID55 set forth in SEQ ID NO:3, wherein the histidine at position 248(H248) is substituted with L, I, V, D, E, F, Y, or W, and wherein thenumbering of the position H248 is based on the numbering of amino acidresidues in SEQ ID NO:1.
 2. An immunogenic composition comprising: a)the variant fHbp according to claim 1; and b) a pharmaceuticallyacceptable excipient.
 3. The immunogenic composition of claim 2, whereinthe variant fHbp is in a vesicle preparation prepared from a Neisseriameningitidis strain.
 4. A method of eliciting an antibody response toNeisseria meningitidis in a mammal, the method comprising administeringto the mammal the immunogenic composition of claim
 2. 5. The method ofclaim 4, wherein the mammal is a human.
 6. A method of eliciting anantibody response to Neisseria meningitidis in a mammal, the methodcomprising administering to the mammal the immunogenic composition ofclaim
 3. 7. The method of claim 6, wherein the mammal is a human.
 8. Thevariant fHbp according to claim 1, comprising the substitution H248L. 9.The immunogenic composition of claim 2, wherein said pharmaceuticallyacceptable excipient comprises an adjuvant.
 10. The immunogeniccomposition of claim 9, wherein the adjuvant is aluminum phosphate oraluminum hydroxide.
 11. The immunogenic composition of claim 9, whereinthe adjuvant is an oil-in-water emulsion.
 12. The immunogeniccomposition of claim 9, wherein the adjuvant is monophosphoryl lipid A.13. The immunogenic composition of claim 9, wherein the adjuvant issaponin.
 14. The immunogenic composition of claim 2, further comprisingNeisserial surface protein A.
 15. An in vitro host cell expressing thevariant fHbp according to claim
 1. 16. The in vitro host cell accordingto claim 15, wherein the host cell is Escherichia coli.
 17. The in vitrohost cell according to claim 15, wherein the host cell is yeast.
 18. Thein vitro host cell according to claim 15, wherein the host cell isNeisseria meningitidis.
 19. The in vitro host cell according to claim18, wherein the Neisseria meningitidis comprises a genetic modificationthat results in decreased or no toxic activity of lipid A.